THE  PRINCIPLES  OF 
IMMUNOLOGY 


THE   PRINCIPLES   OF 
IMMUNOLOGY 


BY 

HOWARD  T.  KARSNER,  M.D. 

PROFESSOR  OF  PATHOLOGY,  WESTERN  RESERVE  UNIVERSITY,  CLEVELAND 

AND 

ENRIQUE  E.  ECKER,  PH.D. 

INSTRUCTOR  IN  IMMUNOLOGY,  WESTERN  RESERVE  UNIVERSITY,  CLEVELAND 


ILLUSTRATED 


PHILADELPHIA  AND  LONDON 
J.  B.  LIPPINCOTT  COMPANY 


'••  • 


(Qf 


COPYKIQHT,  1921,  BY  J.  B.  LIPPINCOTT  COMPANY 


Electrotyped  and  Printed  by  J.  B.  Lippincott  Company 
The  Washington  Square  Press,  Philadelphia,  U.S.A. 


DEDICATED 
TO 

CHARLES  KARSNER 
WILLIAM  C.  KARSNER 
CHARLES  W.  KARSNER 

DANIEL  KARSNER 

AND 

JAMES  H.  M.  KARSNER 
DOCTORS  OP  MEDICINE 


694119 


PREFACE 

THIS  book  has  been  prepared  in  the  hope  that  a  concise  state- 
ment of  the  facts  and  more  important  hypotheses  concerning  resist- 
ance to  infection  may  serve  to  provide  a  clear  understanding  of  a 
subject  of  the  utmost  importance  in  modern  diagnosis  and  treat- 
ment. Designed  primarily  for  students  of  medicine  and  for  those 
practitioners  whose  duties  have  made  it  impossible  to  digest  a  large 
mass  of  publications  on  the  subject,  the  scope  of  the  book  is 
restricted  to  fundamental  principles.  The  plan  throughout  is  to  pre- 
sent on  an  experimental  basis  the  demonstrated  facts  and  to  supple- 
ment these  with  brief  discussions  of  the  practical  and  theoretical 
bearing  of  the  phenomena  upon  resistance  and  disease  in  man.  A 
few  illustrations  have  been  inserted,  but  it  must  be  recognized  that 
technical  details  can  only  be  fully  comprehended  on  the  basis  of 
actual  work  with  the  methods.  The  usual  diagrams  of  the  side- 
chain  theory  have  been  omitted  because  of  the  belief  that  they 
serve  to  confuse  rather  than  clarify  the  conception  of  processes 
whose  fundamental  basis  lies  in  the  field  of  physical  chemistry. 
Certain  material  concerning  the  practical  application  of  immunology 
to  the  prevention  and  cure  of  disease  has  been  collected  in  three 
appendices.  These  have  been  added  in  order  to  explain  the  basis  of  the 
practical  methods  rather  than  as  an  exact  guide  in  their  application. 

Knowledge  progresses  from  the  known  to  the  unknown,  from 
the  simple  to  the  complex,  and  if  the  brevity  of  the  book  serves  to 
implant  essentials  in  such  a  way  that  the  reader  not  only  grasps  the 
facts,  but  finds  himself  stimulated  to  seek  further  information  and 
discussion  in  more  comprehensive  works,  the  most  compelling  aim 
of  this  book  will  have  been  achieved.  For  this  purpose  books 
which  we  have  used  with  considerable  freedom  are  recommended: 
Zinsser,  "  Infection  and  Resistance  " ;  Wells,  "  Chemical  Pathol- 
ogy " ;  Kolmer,  "  Infection,  Immunity,  and  Specific  Therapy " ; 
Kraus  and  Levaditi,  "  Handbuch  der  Technik  und  Methodik  der 
Immunitatsforschung  " ;  Muir,  "  Studies  on  Immunity  " ;  Kolle  and 
Wassermann,  "Handbuch  der  pathogenen  Mikroorganismen " ; 
Metchnikoff,  "  Immunity  in  Infective  Diseases  ";  Bordet,  "  Traite  de 
rimmunite  dans  les  Maladies  Infectieuses  " ;  Besredka,  "  Anaphy- 
laxis  and  Antianaphylaxis " ;  Bordet  and  Gay,  "  Studies  in 
Immunity  " ;  Gay,  "  Typhoid  Fever  " ;  Browning,  "  Applied  Bacteri- 
ology"; Craig,  "The  Wassermann  Test";  Noguchi,  "Serum 
Diagnosis  of  Syphilis  " ;  Zinsser,  Hopkins,  and  Ottenberg,  "  Lab- 
oratory Course  in  Serum  Study."  The  names  of  those  who  have 
contributed  to  the  literature  are  given  in  the  text,  but  precise  refer- 
ences have  been  omitted,  since  the  articles  can  be  found  by  refer- 
ence to  such  bibliographic  journals  as  the  Index  Medicus,  The  Index 

vii 


viii  PREFACE 

Catalogue  of  the  Surgeon  General's  Office,  and  in  particularly  available 
form  in  the  Quarterly  Cumulative  Index  of  the  American  Medical 
Association.  Every  effort  has  been  made  to  give  credit  where  it 
belongs ;  if  omissions  or  errors  have  been  made  they  are  due  to  the 
vast  amount  of  material  that  has  been  accumulated  on  this  subject 
rather  than  to  intentional  oversight. 

Our  thanks  are  due  to  our  colleague,  Doctor  Maurice  L. 
Richardson,  for  extremely  valuable  aid  in  the  revision  of  the  manu- 
script, to  Mr.  E.  L.  Miller  for  three  important  microscopic  draw- 
ings, to  Miss  May  E.  Treter  and  Miss  Catherine  E.  Lennon  for 
faithful  and  painstaking  clerical  work.  Mr.  W.  T.  Brownlow,  of 
Cleveland,  has  made  the  line  drawings  and  Mr.  E.  F.  Faber,  of 
Philadelphia,  the  drawings  of  the  lungs  in  anaphylactic  shock.  We 
have  taken  materials  from  certain  journals  and  make  grateful 
acknowledgment  by  reference  in  the  text. 

January,  1921. 

HOWARD  T.  KARSNER, 
ENRIQUE  E.  ECKER. 


CONTENTS 


CHAPTER.  PAGE 

PREFACE vii 

LIST  OF  ILLUSTRATIONS xi 

INTRODUCTION xiii 

I.  VIRULENCE  OF  ORGANISMS i 

II.  GENERAL  CONDITIONS  OF  INFECTION  AND  RESISTANCE n 

III.  THE  GENERAL  PHENOMENA  OF  IMMUNITY 16 

TYPES  OF  IMMUNITY. 

THEORIES  OF  IMMUNITY. 

SITE  OF  ANTIBODY  FORMATION. 

IV.  TOXINS  AND  ANTITOXINS 37 

BACTERIAL  TOXINS  AND  ANTITOXINS. 

DIPHTHERIA. 

TETANUS. 

DYSENTERY. 

BACILLUS  BOTULINUS. 

GAS  BACILLUS. 

BACTERIAL  HEMOTOXINS 
PHYTOTOXINS. 
ZOOTOXINS. 

V.  AGGLUTININS  AND  PRECIPITINS 78 

BACTERIAL  AGGLUTININS. 

HEMAGGLUTININS. 

PRECIPITINS. 

VI.  CYTOLYSINS 115 

HEMOLYSINS. 
CYTOTOXINS. 
BACTERIOLYSINS. 

VII.  CELLULAR  RESISTANCE 151 

PHAGOCYTOSIS. 

OPSONINS. 
OTHER  FORMS  OF  CELLULAR  RESISTANCE. 

VIII.  COMPLEMENT  FIXATION 173 

THE  BORDET-GENGOU  PHENOMENON. 

IX.  APPLICATION  OF  COMPLEMENT  FIXATION  TO  THE  DIAGNOSIS  OF  DISEASE.  186 
THE  WASSERMANN  REACTION. 
COMPLEMENT  FIXATION  IN  TUBERCULOSIS. 
COMPLEMENT  FIXATION  IN  GONOCOCCUS  INFECTIONS. 
OTHER  COMPLEMENT  FIXATION  TESTS. 

X.  HYPERSUSCEPTIBILITY 208 

ANAPHYLAXIS. 

ANAPHYLACTOID  PHENOMENA. 
RELATION  OF  ANAPHYLAXIS  TO  IMMUNITY. 

XI.  HYPERSUSCEPTIBILITY  IN  MAN 228 

SERUM  DISEASE. 
ANAPHYLACTIC  SHOCK. 
NATURAL  HYPERSUSCEPTIBILITY. 
THE  TUBERCULIN  AND  SIMILAR  TESTS. 

XII.  DEFENSIVE  FERMENTS 245 

THE  ABDERHALDEN  TEST. 

APPENDIX. 

A.  THERAPEUTIC  EMPLOYMENT  OF  BLOOD  SERUM 252 

B.  PROPHYLACTIC  VACCINATION .' 272 

C.  VACCINE  THERAPY 296 


LIST  OF  ILLUSTRATIONS 

FIG.  PAGE 

1 .  Apparatus  for  Filtration  through  Porcelain 43 

2.  The  Rosenau  or  Reichel  Syringe  for  Injecting  Toxin-antitoxin  Mixtures. . .  47 

3.  Wooden  Box  for  Holding  Rabbits 79 

4.  Method  of  Obtaining  Blood  from  the  Rabbit's  Ear 80 

5.  Method  of  Complete  Bleeding  from  the  Femoral  Vessels  of  the  Rabbit.  ...  81 

6.  Collection  of  Serum  in  a  Flask 82 

7.  Method  of  Drawing  Up  Measured  Volumes  of  Fluid  into  a  Graduated  Pipette  83 

8.  The  Wright  Tube  for  Obtaining  Small  Quantities  of  Blood  Serum 85 

9.  Coiled  Pipette  for  Taking  Up  Small  Quantities  of  Fluids 85 

10.  Microscopic  Drawing  of  Bacterial  Agglutination 84 

11.  The  Nipple  Pipette 96 

12.  Hemolysis  in  the  Test  Tube 1 18 

13.  Quantitative  Relations  of  Amboceptor  and  Complement  in  Hemolysis 119 

14.  Method  of  Obtaining  Blood  from  Guinea-pig 128 

15.  Stages  of  Lysis  in  Cholera  Vibrios 144 

16.  Microscopic  Drawing  of  Phagocytosis 154 

17.  Microscopic  Drawing  of  Guinea-pig  Lung  in  Anaphy lactic  Shock 214 

1 8.  Blood-pressure  Tracing  from  Dog  in  Anaphylactic  Shock 216 

PLATES 

PLATE  I.  Positive  Schick  Reaction  of  Moderate  Severity  Seventy-two  Hours 
After  the  Intracutaneous  Injection  of  One-fortieth  the  Minimal 
Lethal  Dose  of  Diphtheria  Toxin.  Patient's  Blood  Serum  Was 

Found  to  Contain  No  Antitoxin 54 

PLATE  II.  Colored  Drawing  of  Guinea-pig  Lungs  in  Anaphylactic  Shock 212 


INTRODUCTION 

THE  history  of  immunology  as  a  science  is  distinctly  modern  and 
in  the  investigation  of  details  dates  back  only  as  far  as  the  time  of 
Louis  Pasteur.  Jenner's  work  on  smallpox  vaccination  represents 
most  painstaking  and  thorough  investigation;  it  was  epochal  in 
character,  and  of  the  utmost  importance  in  practical  results,  but  was 
not  immediately  followed  by  any  general  application  to  other  dis- 
eases, probably  because  of  the  limitations  of  technical  methods. 
Observations  of  the  phenomena  of  immunity  were,  however,  made 
in  ancient  times  and  the  resistance  to  second  attacks  of  such  dis- 
eases as  measles,  scarlatina,  variola,  varicella  must  have  been  com- 
mon knowledge  from  the  earliest  days  of  the  human  race.  Whilst 
many  of  the  earlier  students  of  medicine  recognized  a  certain  simi- 
larity between  poisoning  and  infectious  disease,  yet  Hippocrates 
could  see  no  such  resemblance  and  his  theory  of  the  four  humors 
was  dominant  throughout  the  Middle  Ages.  With  minor  exceptions 
this  belief  held  sway  until  well  into  the  Renaissance.  In  1548,  how- 
ever, Fracastore  proposed  the  theory  that  infection  was  carried 
from  person  to  person  "  per  contactum  "  or  "  per  fomites,"  and  from 
this  time  dates  real  progress  in  the  investigation  of  infectious  dis- 
ease. This  led  subsequently  to  the  establishment  of  two  schools  of 
thought,  the  one  believing  disease  to  be  due  to  substances  of  basic 
or  acid  principle,  and  the  other  believing  disease  to  be  due  to  para- 
sites. The  development  of  the  latter  idea  was  forced  to  await  the 
discovery  of  means  to  view  minute  parasites  and,  as  a  matter  of 
fact,  was  delayed  much  longer,  because  the  invention  of  the  micro- 
scope by  Kircher  in  1659  and  van  Leeuwenhoek  in  1675  far  ante- 
dated the  connection  now  established  between  minute  parasites  and 
infectious  disease.  Nevertheless,  Plenciz  in  1762  expressed  a  belief 
in  the  direct  etiological  connection  between  certain  forms  of  disease 
and  microorganisms,  and  established  the  conception  of  the  "  con- 
tagium  vivum."  This  idea  was  revived  by  Henle  and  by 
Brettoneau,  but  attracted  no  permanent  attention. 

As  perhaps  the  first  observation  leading  up  to  our  present  con- 
ception of  infectious  diseases,  and  therefore  to  immunity  against 
them,  was  the  discovery  in  1837  by  Schwann  that  certain  forms  of 
fermentation  are  due  to  the  presence  of  yeasts,  an  observation  made 
at  about  the  same  time  by  Cagniard-Latour.  Although  at  this  time 
there  was  little,  if  any,  thought  that  this  discovery  had  any  impor- 
tant bearing  on  infectious  disease,  yet  within  the  succeeding  decade 
favus,  thrush,  and  pityriasis  versicolor  had  been  demonstrated  to  be 
due  to  specific  fungi.  Nevertheless,  the  possible  similarity  of  fer- 
mentation and  infectious  disease  had  been  considered  in  a  more  or 
less  philosophical  way,  and  Robert  Boyle  had  said :  "  He  that  thor- 

xiii 


xiv  INTRODUCTION 

oughly  understands  the  nature  of  ferments  and  fermentations  shall 
be  much  better  able  than  he  that  ignores  them  to  give  a  fair  account 
of  diverse  phenomena  of  certain  diseases  (as  well  fevers  as  others), 
which  will  perhaps  be  never  properly  understood  without  an  in- 
sight into  the  doctrine  of  fermentations."  In  the  further  develop- 
ment of  the  origin  of  infectious  disease  in  living  organisms  perhaps 
the  work  of  Rayer  and  Davaine  on  anthrax  was  of  the  utmost  im- 
portance. They  reported  in  1850  that  in  the  blood  of  anthrax  vic- 
tims "  are  found  little  thread-like  bodies  about  twice  the  length  of  a 
blood-corpuscle.  These  little  bodies  exhibit  no  spontaneous  motion." 
In  1863  Davaine  showed  that  the  blood  containing  these  rods  could 
transmit  the  disease  while  blood  free  from  them  did  not  transmit 
the  infectious  agent.  Davaine  suggested  at  this  time  that  the 
manifestations  of  the  disease  might  represent  the  results  of  the 
specific  fermentation  produced  by  these  bacilli.  Such  a  parasitic  concep- 
tion of  disease  was  further  supported  by  the  discovery  in  1873  of  the 
spirillum  of  relapsing  fever  by  Obermeier.  Subsequently  the  work  of 
Louis  Pasteur,  Koch,  and  the  great  school  of  early  bacteriologists  gave 
the  final  evidence  in  support  of  the  "  contagium  vivum." 

Although  the  essential  development  of  the  science  of  immu- 
nology necessarily  awaited  the  critical  study  of  infectious  disease, 
as  can  be  seen  from  the  foregoing  summary  of  the  development  of 
the  knowledge  of  the  cause  of  infections,  yet  throughout  the  ages 
there  had  been  speculations  as  to  the  nature  of  immunity  running 
hand  in  hand  with  hypotheses  as  to  the  nature  of  infection.  Im- 
munology took  its  most  important  step  forward  more  than  a  half 
century  before  the  work  of  Schwann  had  reached  its  fruition  in  the 
studies  of  Davaine,  Obermeier,  and  Pasteur ;  namely,  in  the  master- 
ful experiments  of  Jenner.  It  is  almost  certain  that  for  at  least  a 
century  before  Jenner's  publication  there  had  been  practised,  in  the 
far  and  near  East  as  well  as  in  certain  parts  of  Europe,  including 
England,  the  inoculation  of  smallpox  during  full  health  in  order  to 
produce  a  mild  attack  of  the  disease  and  thus  protect  against  later 
more  severe  or  fatal  attacks.  It  is  indeed  possible,  as  claimed  by 
Carburi,  that  such  a  procedure  originated  in  Europe  as  early  as  the 
sixteenth  century  and  was  carried  to  Constantinople  and  thence  to 
the  far  East.  Similar  attempts  to  produce  mild  attacks  of  other 
diseases  were  tried,  but  with  little  success,  as,  for  example,  the  work 
or  Vesepremi  in  1755  with  plague,  of  Home  in  1757  with  measles,  and 
of  Turenne  in  1844  with  syphilis.  It  seems  unlikely,  however,  that 
any  of  this  work  had  any  direct  bearing  on  the  discovery  of  Jenner. 
Sprengell  states  that  for  many  years  before  Jenner's  time  the  pro- 
tective influence  of  cowpox  against  smallpox  was  known  in  certain 
districts  of  Ireland,  Holstein,  Brandenburg,  Switzerland,  Catalonia, 
Peru,  and  the  East  Indies.  Similar  observations  had  been  published, 
as,  for  example,  the  statement  of  Bose  in  1769,  that  persons  who 
had  suffered  cowpox  were  not  subsequently  attacked  by  smallpox. 
Jesty  in  1774  had  inoculated  some  members  of  his  family  with  cow- 


INTRODUCTION  xv 

pox  and  reported  that  they  remained  free  from  smallpox.  In  1791 
Jensen  and  Plett  practised  protective  inoculation  with  cowpox  and 
reported  good  results,  as  did  also  Penster  in  1765.  None  of  these 
studies,  however,  bore  critical  scientific  examination,  nor  did  they 
serve  to  stimulate  active  work  along  this  line.  Indeed,  it  seems  un- 
likely that  they  influenced  Jenner  in  any  way.  Jenner  brought  to 
bear  the  critical  method  of  the  experimental  investigator  and  proved 
the  point.  The  method  was  rapidly  put  into  clinical  practice, 
spread  over  the  British  Isles  and  Europe  and  stood  the  test  of 
time  and  wide  application.  With  very  slight  modifications  it  stands 
to-day,  in  spite  of  our  great  advances  in  the  study  of  immunity^as 
the  most  effective  method  we  have  to  guard  against  infectious  dis- 
ease. Jenner  vaccinated  a  boy  on  the  arm  with  cowpox  virus  ob- 
tained from  a  lesion  on  the  hand  of  a  dairy  maid,  and  subsequently 
inoculated  the  boy  with  fresh  smallpox  virus,  which  failed  to  pro- 
duce the  disease.  He  also  reported  an  attempt  to  inoculate  small- 
pox unsuccessfully  in  ten  persons  who  had  had  cowpox  nine 
months  to  fifty-three  years  previously.  In  1800  Waterhouse  in 
Boston  repeated  the  experiment  of  Jenner  on  his  own  son,  and  in 
1802  performed  a  more  extensive  and  even  more  critical  experi- 
ment, in  which  he  vaccinated  nineteen  boys  with  cowpox.  Twelve 
were  inoculated  with  smallpox  virus  three  months  later  and  failed 
to  develop  the  disease,  the  same  virus  being  inoculated  at  the  same 
time  into  two  unvaccinated  boys,  producing  well-developed  small- 
pox. The  virus  from  these  latter  two  boys  was  later  inoculated  into 
all  the  iiineteen  vaccinated  boys  without,  results.  Thus  began  the 
period  of  experimental  investigation  of  the  phenomena  of  immunity. 
Further  progress  of  importance  was  not  made  until  1880,  when 
Pasteur  announced  his  results  in  vaccination  against  chicken 
cholera.  No  brief  review  such  as  this  can  do  justice  to  the  stimulus 
to  modern  biological  science  furnished  by  this  man  and  his  asso- 
ciates, and  the  reader  is  referred  to  the  interesting  and  intimate 
view  of  the  life  of  Pasteur  written  by  Valery  Radot,  his  son-in-law. 
At  the  beginning  of  Pasteur's  work  the  theory  of  spontaneous  gen- 
eration was  still  generally  accepted  by  the  scientific  world,  and  be- 
fore he  was  compelled  to  cease  his  active  investigations  not  only 
had  this  theory  been  overthrown,  but  also  the  ideas  of  chemists  in 
regard  to  crystallization  and  to  the  rotation  of  light  by  bodies  in 
solution  had  been  completely  revised,  the  silk  and  wine  industries 
of  France,  and  indeed  of  the  world,  had  been  entirely  rejuvenated, 
the  bacteriological  cause  of  numerous  diseases  conclusively  proven, 
and  the  science  of  immunology  put  on  a  plane  where  its  progress 
must  be  uninterrupted.  His  first  contribution  to  the  science  of  im- 
munology was  in  connection  with  his  work  on  chicken  cholera. 
Although  he  did  not  offer  it  as  such,  nevertheless,  this  incident  well 
illustrated  his  doctrine  that  "  chance  favors  the  prepared  mind."  He 
had  saved  some  old  cultures  of  this  bacterium  and  later  found  that 
they  were  avirulent.  He  subsequently  tried  to  cause  the  disease  in 


xvi  INTRODUCTION 

animals  which  had  been  inoculated  with  this  virus,  using  the  second 
time  a  culture  which  was  virulent  for  untreated  fowl.  He  showed 
that  the  inoculated  fowl  were  immune  to  the  virulent  culture.  In 
1881  he  demonstrated  with  his  collaborators,  Chamberland  and 
Roux,  that  this  was  not  an  isolated  fact,  but  that  essentially  the 
same  thing  had  been  accomplished  with  anthrax.  The  virus  of 
anthrax  could  not  be  attenuated  by  the  same  simple  method  as  for 
fowl  cholera,  because  the  bacillus  anthracis  preserves  its  virulence 
by  the  formation  of  spores.  They  showed,  however,  that  they  could 
prevent  the  formation  of  spores  by  growing  the  bacillus  at  42°  to 
43°  C.  At  this  temperature  growth  of  six  to  eight  days  sufficiently 
attenuates  the  organism  for  protective  inoculation.  The  proof  of 
the  vaccination  was  given  publicly  before  the  Society  of  Agricul- 
ture at  Melun.  For  this  phenomenon  Pasteur  used  the  term  vac- 
cination, and  in  London  in  1881  said:  "  I  have  lent  to  the  expression 
vaccination  an  extension  that  I  hope  science  will  consecrate  as  a 
homage  to  the  merit  and  immense  services  rendered  to  humanity 
by  one  of  the  greatest  men  of  England — Jenner."  In  1882  Pasteur 
and  Loir  confirmed  Thuillier's  observations  on  the  cause  of  swine 
fever  and  then  successfully  vaccinated  pigs  against  this  disease. 
Then  in  1885-1886  came  the  final  brilliant  chapter  in  the  work  with 
rabies,  in  which  vaccination  was  practised  without  definite  knowl- 
edge of  the  etiological  agent.  The  work  with  rabies  was  of  further 
importance  in  that  it  led  to  the  discovery  of  the  fact  that  a  virus 
may  be  increased  in  virulence,  a  phenomenon  quite  the  reverse  of 
the  earlier  discovery  of  the  possibility  of  attenuation. 

In  his  studies  Pasteur  had  worked  almost  entirely  with  the  active 
organisms  causing  disease,  and  the  next  step  forward  was  the  dis- 
covery that  the  products  of  bacterial  growth  and  activity  can  be 
utilized  in  the  development  of  immunity.  Salmon  and  Theobald 
Smith  published  in  1886  their  studies  on  the  immunization  of  hogs 
against  hog  cholera  by  the  use  of  the  products  of  the  specific  organ- 
isms. This  idea  had  been  suggested  by  LoefHer  in  1884,  but  not 
proven.  Before  he  had  made  any  conclusive  experiments  the  sub- 
ject had  been  taken  up  by  numerous  other  investigators.  Behring  and 
Kitasato  in  1890  had  discovered  tetanus  toxin  and  Roux  and  Yersin  in 
1 888- 1 889- 1 890  had  published  their  discovery  of  diphtheria  toxin. 
These  workers  showed  that  the  symptoms  of  the  special  diseases 
studied  could  be  reproduced  by  the  soluble  products  of  the  causative 
organisms  and  by  their  later  work  that  one  of  the  important  phases  of 
immunity  is  due  to  the  development  of  substances  capable  of  neu- 
tralizing these  products.  It  became  evident  with  further  work  that 
this  principle  does  not  apply  to  all  pathogenic  organisms,  and  the 
work  of  Pfeiffer  with  cholera  in  1891  led  to  the  differentiation  of 
exotoxins  and  endotoxins. 

The  antagonistic  action  of  blood  and  body  fluids  on  putrefaction 
had  been  noted  by  John  Hunter,  Traube,  and  Lister,  but  Grohman 
in  1884  was  the  first  to  publish  well-founded  experiments  upon  the 


INTRODUCTION  xvii 

inhibition  by  fresh  plasma  of  the  actual  growth  of  bacteria.  Fliigge 
and  Nuttall  in  1888  demonstrated  under  the  microscope  the  destruc- 
tion of  bacteria  by  blood,  and  Buchner  in  1889  showed  this  property 
to  be  present  in  the  serum.  At  about  the  same  time  the  work  of 
Richet  and  Hericourt  and  of  Babes  and  Lepp  showed  that  an 
immunity  artificially  produced  against  pyogenic  cocci  and  against 
the  virus  of  rabies  could  be  transferred  from  one  animal  to  another 
by  means  of  the  blood  serum.  These  studies  were  followed  almost 
immediately  by  the  discoveries  of  Behring  and  Kitasato  that  the 
serum  of  animals  immunized  to  the  toxins  of  tetanus  and  of  diph- 
theria bacilli  not  only  could  produce  immunity  in  other  animals,  but 
that  the  specific  disease  could  be  cured  by  the  use  of  the  respective 
sera.  These  discoveries  led  immediately  to  the  development  of  serum 
therapy,  and  in  1894  diphtheria  antitoxin  was  being  marketed  in 
Germany.  Contemporaneously  with  these  developments  Metchnikoff 
conducted  his  observations  and  experiments  upon  phagocytosis,  and 
in  1883  published  his  "  Recherches  sur  la  digestion  intracellulaire." 
He  studied  various  lower  forms  of  life,  such  as  echinoderms,  and 
found  that  during  metamorphosis  the  atrophic  cells  of  the  larvae  are 
devoured  by  other  cells,  either  leucocytes  or  other  phagocytic  cells. 
These  studies  were  later  extended  to  include  reparative  conditions, 
such  as  the  healing  of  wounds  and  resistance  to  infection.  The  out- 
come was  a  series  of  brilliant  discoveries  of  the  part  phagocytosis 
plays  in  combating  bacterial  invasion,  and  ultimately  the  practical 
application  in  the  use  of  bacterial  vaccines  for  prevention  and  treat- 
ment of  infectious  disease.  The  discovery  of  the  various  forms  of 
immune  bodies  and  of  the  substances  which  might  lead  to  the  pro- 
duction of  such  immune  bodies  followed  with  considerable  rapidity, 
but  the  details  may  best  be  left  to  the  study  of  the  particular  immune 
bodies  concerned,  which  include  agglutinins  and  precipitins,  cytoly- 
sins,  and  other  complement  binding  substances.  "  That  a  plague 
of  diarrhea  in  a  poultry  yard,  studied  by  a  professor  of  chemistry, 
should  be  the  seed  from  which  has  grown  the  vast  development  of 
later  years  is  a  strange  fact,  but  fact,  nevertheless  "  (Adami). 


THE   PRINCIPLES   OF 
IMMUNOLOGY 

CHAPTER  I 
VIRULENCE  OF  ORGANISMS 

MUTUAL  RELATIONS  OF  PARASITE  AND  HOST. 
PARASITISM. 

VIRULENCE. 

METHOD  OF  DEMONSTRATION. 
THE   BASIS    OF   VIRULENCE. 
ANIMAL  PASSAGE. 

CAPSULE  FORMATION. 
AGGRESSINS. 

POISONOUS    SUBSTANCES    OF   BACTERIAL   ORIGIN. 
PTOMAINS. 

TRUE  TOXINS    (EXOTOXINS). 
ENDOTOXINS. 

POISONOUS  BACTERIAL  PROTEINS. 
ALTERATIONS   OF  VIRULENCE. 
INCREASE   OF   VIRULENCE. 
DECREASE  OF  VIRULENCE. 

Mutual  Relations  of  Host  and  Parasite. — The  existence  of  in- 
fectious disease  depends  fundamentally  upon  the  invasion  of  a 
plant  or  animal  by  some  infective  agent.  The  infective  agent  is 
usually  a  microorganism  either  bacterial  or  protozoan  in  nature, 
although  infestations  by  larger  organisms,  such  as  worms  within 
the  body  or  various  forms  of  pediculi  upon  the  surface,  are  some- 
times spoken  of  as  infections.  In  addition  to  bacteria  the  vegetable 
world  includes  parasites,  such  as  yeasts  and  fungi,  which  are  cap- 
able of  producing  disease.  The  actual  production  of  disease  depends 
fundamentally  upon  the  interrelationship  between  the  infectious 
agent  and  the  invaded  body.  Bacteria  are  widely  distributed  in 
nature,  but  the  greater  number  of  varieties  have  no  capacity  for  the 
production  of  disease.  Those  which  produce  disease  are  spoken  of 
as  pathogenic,  and  those  which  do  not  produce  disease  are  spoken 
of  as  non-pathogenic.  There  are  forms,  however,  which  although 
they  ordinarily  do  not  produce  disease,  may,  under  certain  circum- 
stances, develop  this  character.  Animals  and  plants  possess  cer- 
tain factors  of  resistance  to  the  invasion  of  pathogenic  organisms, 
and  the  pathogenic  organisms  possess  certain  characters  which 
favor  invasion.  Both  animals  and  plants  live  in  constant  associa- 
tion with  microorganisms,  and  apparently  in  many  instances  both 
are  benefited  by  this  association.  It  is  well  known  that  certain 
plants  require  for  favorable  development  the  association  of  the  nitri- 
fying bacteria.  The  intestinal  canal  of  man,  although  free  from 


.  V  :  ;liRE  PRINCIPLES  OF  IMMUNOLOGY 


few  days  of  extra-uterine  life,  soon  becomes  in- 
habited by  large  numbers  of  organisms,  which  produce  no  deleteri- 
ous effect  under  ordinary  circumstances  and,  in  fact,  appear  to  aid 
in  the  process  of  digestion.  Animals  may  adapt  themselves  to 
organisms  even  of  the  pathogenic  varieties,  as,  for  example,  in  the 
condition  known  as  the  "  carrier  state,"  in  which  virulent  diphtheria 
bacilli  or  virulent  typhoid  bacilli  are  harbored  without  apparent 
harm.  This  capacity  is  due  to  certain  changes  which  take 
place  in  the  body,  so  that  the  organisms  and  their  products  do  no 
damage.  In  the  carrier  state  the  organisms  themselves  have  prob- 
ably developed  a  state  of  resistance  against  substances  produced  in 
the  host  which  ordinarily  would  destroy  the  organisms  and  neu- 
tralize their  toxic  products. 

Parasitism. — The  parasite  is  a  living  organism  which  carries  on 
its  existence  within  or  upon  its  host,  and  derives  its  nutrition  there- 
from. Parasitic  bacteria  include  both  pathogenic  and  non-pathogenic 
forms.  Bail  has  classified  bacteria  in  three  forms:  (i)  Pure  sapro- 
phytes, which  do  not  develop  within  living  animal  tissue,  but  derive 
nutrition  from  dead  material;  these  may  be  pathogenic,  provided 
they  produce  poisonous  substances  which  may  be  absorbed,  as  is 
the  case  with  the  bacillus  aerogenes  capsulatus;  (2)  pure  parasites, 
which  live  entirely  within  tissues,  including  such  organisms  as  the 
anthrax  bacillus ;  they  may  exist  in  a  vegetative  form  for  long 
periods  of  time  outside  the  body;  (3)  half  parasites,  which  may  be 
pathogenic  if  introduced  into  the  animal  body,  but  do  not  possess 
the  invasive  character  and  the  necessity  for  life  within  tissues  ex- 
hibited by  the  pure  parasites.  Most  of  the  bacteria  pathogenic  for 
man  belong  in  this  last  group,  as,  for  example,  the  bacillus  typhosus 
and  the  cholera  vibrio.  Such  organisms  as  these  may  live  and  grow 
for  long  periods  of  time  in  water,  and  in  foods  of  various  kinds,  may 
vegetate  for  a  certain  period  under  unfavorable  conditions,  but  upon 
introduction  into  a  susceptible  host  produce  local  lesions  and  in 
some  instances  may  become  moderately  invasive  for  the  entire 
organism.  Symbiosis  has  relatively  little  significance  in  human 
medicine,  but  certain  instances  occur,  as,  for  example,  the  apparent 
symbiosis  of  the  fusiform  bacillus  and  the  spirillum  of  Vincent's 
angina.  Certain  parasitic  protozoa,  such  as  the  endameba  histolytica 
of  dysentery,  require  the  associated  presence  of  bacteria,  but  these 
latter  are  not  necessarily  pathogenic  and  the  phenomenon  is  not 
that  of  symbiosis,  because  the  endamebse  live  at  the  expense  of  the 
bacteria,  and  the  organisms,  therefore,  are  not  mutually  advantage- 
ous to  the  existence  of  each  other. 

Virulence. — By  the  term  virulence  is  indicated  the  capacity  of 
an  organism  to  produce  disease.  The  degree  of  virulence  may  differ, 
not  only  between  different  species  of  organisms,  but  between  strains 
within  the  same  species.  It  is  probably  true  also  that  individual 
organisms  in  the  same  culture  possess  different  degrees  of  virulence. 
Furthermore,  the  virulence  of  a  species  or  of  a  particular  strain 


VIRULENCE  OF  ORGANISMS  3 

may  be  altered  by  favorable  or  unfavorable  conditions.  Virulence, 
however,  does  not  depend  entirely  upon  characters  inherent  in  the 
infectious  agent,  because  the  production  of  disease  is  an  exhibition 
of  reaction  between  invading  organism  and  host.  We  may,  there- 
fore, say  that  virulence  depends  upon  two  groups  of  factors,  those 
inherent  in  the  invading  organism  and  those  dependent  upon  the 
resistance  exhibited  by  the  attacked  individual.  This  resistance  on 
the  part  of  the  host  is  represented  by  the  condition  of  immunity  and 
will  be  discussed  subsequently.  The  capacity  of  the  infecting  organ- 
ism in  the  production  of  disease  depends  upon  certain  inherent  ele- 
ments of  virulence  which  are  not  well  understood,  upon  the  capacity 
of  the  organism  to  protect  itself  against  the  defensive  mechanism  of 
the  host,  upon  the  capacity  to  produce  certain  substances  which  aid 
invasion,  and  upon  the  development  of  poisonous  bacterial  products. 

Demonstration  of  Virulence. — Inherent  virulence  of  organisms 
may  be  demonstrated  by  the  administration  of  accurately  measured 
doses  of  the  organism  and  observance  of  the  effects  upon  susceptible 
animals.  Ordinarily  the  dose  is  measured  in  the  form  of  certain 
quantities  of  fluid  culture.  Growths  on  solid  media  may  be  meas- 
ured by  the  use  of  a  platinum  loop  so  standardized  as  to  take  up 
approximately  2  mg.  of  the  organisms.  Such  growths  may  also  be 
measured  by  suspension  in  a  suitable  menstruum.  If  5.0  c.c.  of  salt 
solution  are  added  to  a  slant  agar  culture,  fractions  of  the  resulting 
5.0  c.c.  suspension  contain  equivalent  fractions  of  the  total  surface 
growth.  The  most  accurate  method  is  that  of  Barber,  who  has  developed 
a  technic  in  which  the  use  of  a  capillary  tube  permits  picking  a  single 
organism  out  of  a  suspension.  Of  importance  in  considering  viru- 
lence from  this  point  of  view  is  not  only  the  quantity  of  organisms 
injected,  but  also  the  length  of  time  they  have  lived  upon  artificial 
media,  inasmuch  as  prolonged  cultivation  leads  to  deterioration  of 
virulence.  If  a  culture  is  maintained  for  a  period  of  time  without 
transplantation  considerable  numbers  of  the  organisms  die,  and 
therefore  may  constitute  a  part  of  the  bulk  injected,  at  the  expense 
of  living  organisms.  This  is  not  a  true  decrease  of  virulence  and 
constitutes  a  factor  of  error.  The  route  of  injection  is  also  of  im- 
portance, because  certain  organisms  may  be  virulent  by  one  route 
of  injection  and  not  so  by  others.  For  example,  the  cholera  vibrio 
may  produce  disease  by  introduction  into  the  intestinal  tract  and  is 
entirely  without  pathogenic  effect  when  introduced  subcutaneously. 

The  Basis  of  Virulence. — The  studies  of  pathogenic  bacteria  have 
shown  that  they  may  acquire  or  in  certain  instances  may  lose  viru- 
lence by  passage  through  animals,  and  that  they  may  lose  virulence 
by  cultivation  upon  artificial  media.  The  method  whereby  they 
acquire  virulence  has  been  extensively  studied.  It  is  well  known 
that  the  pneumococcus  possesses  a  capsule  when  growing  in  ani- 
mal tissue,  but  that  it  loses  its  capsule  after  artificial  cultivation. 
This  is  true  of  certain  other  organisms,  and  it  has  been  demon- 
strated that  the  protection  afforded  by  the  capsule  makes  these 


4  THE  PRINCIPLES  OF  IMMUNOLOGY 

organisms  resistant  to  the  defensive  phenomena,  phagocytosis  and 
agglutination,  as  will  be  discussed  in  subsequent  chapters.  There- 
fore, capsule  formation  may  well  constitute  an  aid  to  invasion. 

Aggressins. — It  was  found  by  Koch  that  tuberculous  animals 
injected  intraperitoneally  with  fresh  cultures  of  tubercle  bacilli  suc- 
cumb soon  after  the  injection,  and  that  a  considerable  amount  of 
exudate  appears  in  the  peritoneum.  This  phenomenon  seems  to 
have  been  the  basis  of  Bail's  aggressin  theory.  Bail  injected  tubercle 
bacilli,  together  with  sterile  tuberculous  exudate,  into  healthy 
guinea-pigs  and  found  that  the  injected  animals  died  in  the  course 
of  twenty-four  hours,  while  control  animals  inoculated  with  the 
exudate  alone  did  not  show  any  appreciable  effect,  and  control 
animals  which  received  tubercle  bacilli  alone  died  only  after  the 
lapse  of  several  weeks.  He  argued  from  this  that  the  sterile  exudate 
must  contain  a  substance  or  substances  which  are  responsible  for  the 
increased  virulence  or  aggressiveness  of  the  bacilli.  He  named  this 
substance  "  aggressin  "  and  believed  that  during  an  infection  the 
organisms  secrete  certain  substances  which  have  power  to  inhibit 
or  destroy  the  protective  powers  of  the  host.  These  bodies  are  sup- 
posed to  be  formed  by  the  living  bacteria  in  the  living  body  only, 
and  the  pathogenicity  of  bacteria  is  said  to  depend,  in  part  at  least, 
upon  their  ability  to  produce  aggressins.  Bail  believed  further  that 
the  germicidal  activity  of  body  fluids  in  natural  immunity  had  been 
overemphasized.  He  had  noted  with  Petterson  that  animals  highly 
susceptible  to  anthrax  often  possessed  sera  which  had  marked  bac- 
tericidal powers  against  the  anthrax  bacillus.  If  such  animals  were 
inoculated  with  a  few  hundred  organisms,  a  number  easily  destroyed 
by  their  sera,  they  nevertheless  rapidly  succumbed  to  the  disease. 
Bail  also  showed  that  the  peritoneal  fluid  of  guinea-pigs  dying  after 
a  fatal  injection  of  typhoid  or  cholera  organisms  possessed  the 
ability  to  increase  the  virulence  or  infectivity  of  particular  strains 
that  would  otherwise  have  been  harmless.  Experiments  of  this 
kind  were  also  performed  in  dysentery,  chicken  cholera,  pneumonia, 
and  staphylococcus  infections,  and  the  results  obtained  were  identi- 
cal with  those  observed  in  the  case  of  tubercle  bacillus.  Heating 
the  exudate  to  60°  C.  instead  of  inhibiting,  increased  the  aggres- 
siveness of  the  organisms.  Small  doses  appeared  to  act  relatively 
more  strongly  than  larger  doses.  In  tuberculous  animals  the  tissues 
seemed  to  be  saturated  with  this  body,  and  when  fluid  collected  in 
the  body  cavities,  as  happens  on  injection  of  tubercle  bacilli,  these 
fluids  contained  large  quantities  of  aggressins  capable  of  inhibiting 
phagocytosis  by  preventing  the  migration  of  polymorphonuclear 
leucocytes.  Bail,  however,  was  not  the  first  to  observe  this  par- 
ticular phase  of  bacterial  offense.  Salmon  and  Smith,  as  early  as 
1884,  noted  that  bacteria  multiply  in  the  tissues  of  their  host  be- 
cause of  a  poisonous  principle  which  is  produced  during  their 
growth  and  multiplication.  Kruse  maintained  that  the  organisms 
secrete  ferment-like  bodies  (referred  to  as  "  lysins  ")  which  have 


VIRULENCE  OF  ORGANISMS  5 

the  power  of  inhibiting  the  bactericidal  activity  of  the  blood  serum, 
thus  allowing  the  invader  opportunity  for  further  invasion.  By  re- 
peated injections  of  aggressin  exudates  into  animals,  Bail  suc- 
ceeded in  immunizing  these  animals  against  various  infections,  thus 
producing  anti-aggressins.  These  rendered  the  bacteria  defenseless 
and  permitted  unhindered  phagocytosis.  The  agglutinative  power 
of  the  sera  of  such  animals  was  markedly  enhanced. 

Wassermann  and  Citron,  and  many  others,  soon  opposed  Bail's 
aggressin  hypothesis,  by  pointing  out  that  the  phenomenon  can  be 
explained  without  assuming  that  a  new  type  of  immune  body  is 
concerned.  Wassermann  and  Citron,  Wolff,  Sauerbeck,  and  also 
Doerr  found  that  the  action  of  the  so-called  aggressins  can  be  ex- 
plained by  the  fact  that  exudates  contain  extracts  of  the  bacteria. 
Artificial  aggressins  were  prepared  by  making  extracts  of  bacteria 
in  vitro.  It  thus  seems  probable  that  the  aggressins  are  nothing 
more  than  endotoxins  which  have  a  negative  chemotatic  influence 
and  a  non-specific  action.  Citron  was  able  to  show  by  means  of  com- 
plement-fixation that  the  exudates  contain  free  bacterial  receptors, 
which  by  absorbing  immune  bodies,  tend  to  neutralize  the  destruc- 
tive power  of  these  antibodies.  Levy  and  Fornet  showed  that  fresh 
twenty-four-  to  forty-eight-hour  culture  filtrates  of  bacillus 
typhosus,  paratyphosus,  pyocyaneus  and  proteus  possess  non- 
specific aggressive  powers  and,  according  to  Ikomikoff,  aggressins 
of  bacillus  coli,  staphylococci,  and  vibrios  will  act  interchangeably, 
thus  showing  the  non-specific  nature  of  these  substances.  From 
Zinsser  and  Dwyer's  experiments  these  bodies  appear  to  be  practi- 
cally identical  with  anaphylatoxins  (see  page  218).  The  addition  of  ana- 
phylatoxin  to  bacteria  will  change  a  sublethal  dose  into  a  lethal  dose. 

Closely  related  to  the  aggressins  are  the  "  virulins  "  of  Rosenow. 
This  author  found  that  freshly  isolated  cultures  of  pneumococci 
were  not  readily  phagocyted,  but  this  property  was  lost  on  repeated 
subculture.  He  prepared  salt  solution  extracts  of  the  virulent 
strains.  Upon  treating  avirulent  strains  for  twenty-four  hours  or 
more  with  these  extracts,  the  avirulent  organisms  became  virulent 
for  animals,  and  at  the  same  time  resistant  to  phagocytosis.  The 
substance  contained  in  the  salt  solution  extracts  capable  of  render- 
ing the  organisms  virulent  was  named  virulin.  This  substance  ap- 
pears to  be  essentially  the  same  as  the  aggressin  prepared  in  vitro 
by  Wassermann  and  Citron.  In  our  opinion,  these  extracts,  whether 
prepared  in  the  form  of  exudates  or  as  extracts,  contain  poisonous 
bodies  which  augment  the  invasiveness  of  the  organism.  They  may 
be  non-specific  bacterial  proteins  or  protein  products,  such  as  are 
probably  contained  in  so-called  anaphylatoxin.  They  may  be 
in  part  endotoxins.  The  anti-aggressins  of  Bail  are  agglutina- 
tive and  are  probably  called  forth  by  the  injection  of  the  ex- 
tracted bacterial  proteins  in  the  exudates  or  extracts.  It  seems 
probable  also  that  the  effect  of  these  anti-aggressins  may  depend 
upon  their  agglutinative  capacity.  The  subject  is  confused  and  in- 


6  THE  PRINCIPLES  OF  IMMUNOLOGY 

tricate,  and  whilst  at  present  we  are  disposed  to  regard  the  aggressins 
as  extracts  of  the  bacterial  proteins  and  their  split  products,  as  well, 
perhaps,  as  exotoxic  in  nature,  further  study  may  offer  more  com- 
plete and  satisfactory  explanation  of  the  problem. 

Production  of  Poisonous  Substances.— The  virulence  of  organ- 
isms depends,  to  a  certain  extent,  upon  the  poisonous  substances  which 
they  produce.  Nevertheless,  virulence  is  not  necessarily  parallel  to 
the  capacity  for  production  of  these  toxic  substances.  The  poison- 
ous bacterial  products  may  be  divided  into  four  groups,  namely,  the 
ptomains,  which  are  the  result  of  decomposition  of  the  media  upon 
which  the  bacteria  grow;  the  exotoxms  or  true  toxins,  which  are 
soluble  poisons  produced  by  the  life  activities  of  the  bacteria  and 
easily  absorbed  and  diffused  in  the  body  of  the  host ;  the  endotox'ms, 
which  develop  within  the  bodies  of  the  bacteria  and  are  liberated 
probably  only  upon  the  death  and  disintegration  of  the  bacteria ;  and 
poisonous  bacterial  proteins,  which  result  in  large  part  from  the  break- 
ing down  of  the  protein  molecules  which  go  to  constitute  the 
bacterial  substance. 

The  ptomains  are  formed  from  the  decomposition  of  the  media 
upon  which  bacteria  grow,  provided  these  media  are  nitrogenous  in 
nature.  The  ptomains  are  basic  substances  formed  not  from  the 
bacteria  themselves,  but  from  the  decomposition  products  of  those 
media  which  contain  nitrogenous  material,  especially  proteins  whose 
nitrogen  is  in  the  form  of  amino-acids.  Most  ptomains  are  combina- 
tions simply  of  carbon,  hydrogen,  and  nitrogen,  and  they  may  be 
divided  into  three  groups,  the  first  of  which  includes  methylamine, 
dimethylamine,  and  trimethylamine ;  the  second  group  somewhat 
more  complex,  contains  putrescin  and  cadavarin;  the  third  group, 
the  so-called  cholin  group,  contains,  in  addition  to  cholin,  neurin, 
muscarin,  and  betain.  The  cholin  group  are  derivatives  of  lecithin. 
Cholin  itself  is  found  in  extremely  minute  amounts  in  body  cells  and 
has  a  relatively  low  degree  of  toxicity.  It  is  a  substance  which  has 
been  the  subject  of  much  experiment  and  hypothesis,  but  there  is 
no  very  good  reason  for  believing  that  it  has  any  great  pathologic 
importance.  Neurin  may  be  transformed  from  cholin,  and  although 
somewhat  similar  chemically,  it  is  highly  poisonous.  Muscarin  is  a 
crystalline  alkaloid  obtained  from  poisonous  mushrooms,  but  is  also 
formed  by  the  decomposition  of  fish;  its  chemical  composition  is 
very  closely  similar  to  that  of  neurin,  and  it  may  be  prepared  syn- 
thetically from  cholin.  Both  neurin  and  muscarin  produce  definite 
toxic  symptoms  in  man  following  subcutaneous  injection  of  i  to  3 
milligrams,  but  when  given  by  mouth  approximately  ten  times  this 
amount  are  required,  indicating  that  probably  the  liver  breaks  up 
and  detoxifies  that  which  is  absorbed  from  the  intestine.  Betain  is 
a  constituent  of  plant  tissues  and  has  a  toxicity  from  one-tenth  to 
one-twentieth  that  of  neurin  and  muscarin.  The  simpler  ptomains 
are  not  extremely  toxic.  The  ptomains  as  a  group  are  not  specific  in 
any  sense,  except  in  so  far  as  they  are  dependent  on  the  chemical 


VIRULENCE  OF  ORGANISMS  7 

composition  of  the  media  upon  which  the  bacteria  grow,  and  any 
differences  in  constitution  of  ptomains  are  differences  due  to  varia- 
tions in  medium  rather  than  variations  of  bacteria.  In  this  respect 
they  differ  from  toxins.  Furthermore,  it  is  not  possible  to  produce 
immune  substances  against  ptomains.  Ptomains  are  not  to  be  con- 
fused with  toxins  produced  by  bacillus  botulinus,  by  bacillus  enteri- 
ditis,  or  other  members  of  the  "  food-poisoning  "  group,  which  are 
true  toxins  and  are  capable  of  inducing  the  formation  of  antitoxins. 
Food  poisoning  may,  therefore,  be  due  to  the  decomposition  of  food 
with  the  production  of  ptomains  which  are  absorbed  and  produce 
toxic  symptoms,  or  may  be  due  to  the  presence  in  food  of  toxins 
produced  by  the  bacillus  botulinus  and  similar  organisms.  In  addi- 
tion to  the  ptomains  which  contain  C,  H  and  N,  a  fourth  group 
contains  also  oxygen,  as  exemplified  in  the  substance  sepsin  ob- 
tained from  decomposing  yeast  cells.  This  is  closely  related  to 
cadavarin  in  its  chemical  composition  and  acts  as  a  powerful  dilator 
of  intestinal  capillary  blood-vessels  from  which  diapedesis  may  occur. 

The  true  toxins  or  exotoxins  are  soluble  and  diffusible  poisonous 
substances  produced  by  the  life  activity  of  bacteria.  They  may  be 
produced  when  the  organisms  exist  in  a  parasitic  state  or  when  they 
grow  upon  artificial  media  and  the  nature  of  a  toxin  for  any  given 
species  is  not  determined  by  the  medium  upon  which  the  organisms 
grow,  except  in  so  far  as  certain  media  favor  the  production  of 
greater  amounts  of  toxin  than  do  others.  The  diphtheria  bacillus 
produces  the  same  toxin  regardless  of  the  medium  upon  which  it  is 
grown,  although  nutrient  veal  broth  is  the  most  favorable  for  toxin 
formation.  The  same  general  statement  is  true  of  the  tetanus 
bacillus  and  those  other  organisms  which  produce  toxins.  Toxins 
are  unlike  ptomains  in  that  they  have  not  a  definite  chemical  com- 
position and  in  that  they  serve  to  induce  antitoxin  formation.  They 
have  certain  resemblances  to  enzymes,  but  are  probably  not  identi- 
cal with  enzymes.  The  nature  of  toxins,  their  action,  and  other 
details  are  considered  in  the  chapter  on  toxins  and  antitoxins. 

The  endotoxins  develop  within  the  bodies  of  bacteria  and  are  not 
secreted  into  the  surrounding  medium.  They  apparently  are  only 
liberated  upon  the  death  and  disintegration  of  the  organisms.  It  is 
not  certain  that  they  can  be  differentiated  absolutely  from  the 
poisonous  bacterial  proteins,  and  it  is  extremely  difficult  to  induce 
antitoxin  formation  by  their  use.  If  they  are  injected  into  an  ani- 
mal the  animal  may  produce  agglutinins  and  precipitins,  but  not 
antitoxin.  This  subject  also  is  discussed  subsequently. 

Poisonous  Bacterial  Proteins. — The  whole  protein  of  certain 
bacteria  is  poisonous,  and  the  work  of  Vaughan  and  Novy  shows 
that  the  split  products  of  bacterial  proteins  produced  by  treatment 
with  alkalinized  alcohol  are  extremely  toxic.  These  substances 
apparently  are  not  specific  as  regards  the  bacteria  from  which  they 
originate,  but  owing  to  their  poisonous  properties  they  may  add  to 
the  virulence  of  the  organisms.  Similarly  toxic  split  products  may 


8  THE  PRINCIPLES  OF  IMMUNOLOGY 

be  obtained  from  other  proteins,  such  as  those  of  cheese  and  milk. 
The  poisonous  effect  is  in  some  way  connected  with  the  foreign 
character  of  proteins.  In  some  respects  these  substances  resemble 
ptomains,  but  they  are  certainly  not  of  the  same  constitution.  They 
are  obtained  from  bacteria  regardless  of  whether  these  produce 
toxins,  endotoxins,  or  ptomains,  and  are  fatal  for  animals  in  very 
short  periods  of  time.  The  methods  for  the  production  of  endo- 
toxins are  such  as  may  lead  to  splitting  of  bacterial  proteins,  and  at 
the  present  time  no  satisfactory  differentiation  can  be  made.  The 
chapter  on  anaphylaxis  and  hypersusceptibility  will  present  a  dis- 
cussion of  the  poisonous  substance  called  anaphylatoxin,  which  may 
also  be  related  to  the  general  group  of  poisonous  split  products. 
The  influence  of  toxin  on  invasion  by  certain  bacteria  is  illustrated 
by  the  recent  work  of  Bullock  and  Cramer.  They  found  that 
bacillus  aerogenes  capsulatus,  vibrion  septique,  bacillus  edematiens, 
and  often  bacillus  tetani,  when  completely  freed  of  toxin  by  washing 
or  by  heating  to  80°  C.  for  one-half  hour  do  not  produce  the  special 
disease  upon  injection  into  the  rat  or  guinea-pig.  The  usual  de- 
fenses of  the  animal,  such  as  bacteriolysis  and  phagocytosis,  are 
sufficient  to  rid  it  of  the  bacteria  in  the  absence  of  toxins.  A  point 
of  further  interest  in  this  work  is  the  discovery  that  if  a  small  dose 
of  a  soluble  ionizable  calcium  salt  be  injected  before  or  at  the  same 
time  as  the  spores  or  toxin-free  bacteria,  the  defenses  are  broken 
down  and  the  special  disease  results.  The  experiments  showed  that 
this  is  not  the  result  of  action  upon  the  bacteria,  but  is  due  rather 
to  some  influence  upon  the  host.  Bullock  and  Cramer  suggest  the 
name  "  cataphylaxis  "  for  the  rupture  of  defense.  Other  salts  have 
no  such  effect,  and  it  is  possible  to  demonstrate  the  antagonistic 
action  of  magnesium  upon  calcium  in  similar  experiments.  It  is 
difficult  to  find  a  series  of  experiments  showing  more  clearly  the 
delicacy  of  balance  between  resistance  and  infection. 

Alterations  of  Virulence — Increase  of  Virulence. — As  has  been 
indicated  above,  virulence  may  be  increased  by  the  passage  of  organ- 
isms through  animals,  and  this  method  is  commonly  employed  in 
laboratory  work.  The  increase  of  virulence  of  the  pneumococcus  by 
passage  through  mice  is  an  excellent  example  of  the  process.  The 
organisms  are  injected  intraperitoneally,  recovered  upon  the  death 
of  the  animal,  cultivated  for  twenty-four  hours,  reinoculated,  and  the 
process  repeated  until  a  satisfactory  degree  of  virulence  is  obtained. 
The  degree  of  virulence  is  usually  measured  in  terms  of  the  bulk  of 
broth  culture  which  will  kill  an  animal  in  a  given  period  of  time. 
In  the  case  of  some  bacteria  an  increase  of  virulence  by  animal 
passage  is  only  effective  for  the  animal  concerned;  and  the  fact 
that  an  organism  exhibits  increased  virulence  for  a  guinea-pig  does 
not  necessarily  presuppose  that  the  same  increase  will  apply  to  other 
animals.  Not  only  is  this  true  of  direct  animal  passage  but,  as  has 
been  shown  by  Danysz,  cultivation  of  an  organism  upon  media  con- 
taining rat  tissue  may  increase  the  virulence  for  the  rat  but  not  for 


VIRULENCE  OF  ORGANISMS  9 

other  animals.  The  importance  of  proper  selection  of  the  animal 
species  for  increasing  bacterial  virulence  is  emphasized  by  the  work 
of  Hussy,  who  found  that  the  passage  of  streptococci  through 
mammals  and  fish  increased  the  virulence,  but  their  passage  through 
birds  decreased  the  virulence.  In  the  increase  of  virulence  by  means 
of  animal  passage,  the  organism  apparently  may  develop  a  mechan- 
ism of  resistance  against  the  protective  activity  of  the  animal  body, 
as  has  been  discussed  above.  Another  method  of  increasing  virulence 
is  to  place  the  organism  in  collodion  sacks.  These  are  planted  in 
the  peritoneal  cavity  of  an  animal  and  apparently  the  slow  diffu- 
sion of  the  animal  fluids  into  the  sack  permits  the  organism  to 
acquire  resistance  to  the  antagonistic  substances  of  the  animal  and 
thus  increases  its  virulence.  A  third  method  of  increasing  virulence 
is  to  grow  organisms  upon  media  which  contain  blood  serum  or 
other  animal  fluids.  By  several  transfers  upon  such  media  the 
organisms  may  acquire  resistance  similar  to  that  obtained  in  the 
other  methods.  A  fourth  method  has  been  applied,  depending  upon 
the  separation  of  the  more  virulent  individuals  in  a  culture  from 
the  less  virulent  at  the  height  of  phagocytosis.  A  culture  is  in- 
oculated into  the  peritoneal  cavity  of  an  animal,  such  as  the  guinea- 
pig,  and  by  removing  small  quantities  at  regular  intervals  the  time 
of  greatest  phagocytosis  by  the  peritoneal  cells  is  determined.  The 
entire  exudate  is  then  withdrawn  and  slowly  centrifuged,  so  as  to 
throw  down  the  cells,  leaving  the  unphagocyted  bacteria  in  the 
supernatant  fluid.  The  organisms  in  the  supernatant  fluids  are 
cultivated,  and  if  they  are  not  sufficiently  virulent  the  process  may 
be  repeated  until  a  satisfactory  culture  is  obtained. 

Decrease  of  Virulence. — The  virulence  of  pathogenic  organisms 
may  be  decreased  by  removing  them  from  the  favorable  environ- 
ment of  the  animal  host  and  growing  them  upon  artificial  culture 
media.  As  they  become  accustomed  to  this  type  of  existence  they 
usually  lose  considerably  in  virulence.  As  has  been  indicated  above, 
there  are  instances  where  animal  passage  may  decrease  the  virulence 
of  certain  infective  agents.  Whereas  the  virus  of  rabies  increases 
up  to  a  standard  maximum  on  passage  through  rabbits,  similar  pas- 
sage through  monkeys  will  decrease  its  virulence.  It  is  probable, 
also,  that  the  natural  passage  from  dog  to  dog  decreases  virulence. 
It  is  now  generally  accepted  that  cowpox  is  the  same  disease  as 
smallpox,  yet  the  inoculation  of  cowpox  into  man  produces  a  very 
mild  form  of  disease.  Therefore,  it  is  to  be  presumed  that  the  pas- 
sage of  smallpox  virus  through  the  calf  reduces  the  virulence.  A 
similar  example  is  found  in  the  work  of  Hussy  on  the  streptococcus 
quoted  above.  Decrease  of  virulence  by  animal  passage  is  not 
clearly  understood.  It  may  be  due  to  the  same  factors  that  influ- 
ence virulence  in  artificial  culture  media,  whereby  the  organisms  in 
an  unfavorable  environment  lose  their  ability  to  combat  the  resist- 
ance of  the  animal  host,  or  it  may  be  due  to  a  direct  lowering  of 
virulence  as  the  result  of  more  or  less  successful  attacks  of  the  pro- 


io  THE  PRINCIPLES  OF  IMMUNOLOGY 

tective  mechanism  of  the  animal  body.  The  influence  of  heat  on  the 
virulence  of  organisms  is  now  well  known.  The  degree  of  heat  and 
the  time  of  exposure  must  be  so  adjusted  as  to  reduce  virulence 
without  causing  actual  death  of  the  organisms.  Similar  reduction 
of  virulence  or  attenuation  may  be  accomplished  by  growing  the 
organisms  at  temperatures  which  are  not  optimal.  The  first  ex- 
ample of  this  was  Pasteur's  work  in  the  attenuation  of  anthrax 
cultures  by  growth  at  42°  to  43°  C.  The  attenuation  by  means  of 
drying  was  practised  in  the  classical  work  of  Pasteur  on  rabies. 
The  virus  contained  in  the  spinal  cord  of  rabbits  was  subjected  to 
desiccation,  and  it  was  found  that  the  longer  the  time  of  desiccation 
the  less  potent  was  the  virus.  Chemical  agents,  such  as  phenol, 
acids,  iodine  and  its  salts,  potassium  bichromate,  and  others  may 
also  be  used  in  proper  concentrations  and  for  proper  periods  of  time 
to  produce  attenuation.  Physical  agencies,  such  as  growth  under 
pressure,  the  influence  of  light,  etc.,  have  been  employed  for  pur- 
poses of  attenuation.  Of  interest  in  connection  with  attenuation  is 
the  fact  that  certain  organisms,  when  introduced  into  the  body,  vary 
in  virulence,  depending  upon  the  route  of  introduction.  For  ex- 
ample, the  virus  of  rabies  may  be  injected  intravenously  into  goats 
and  sheep  without  producing  rabies.  This  injection,  however,  serves 
to  confer  a  certain  degree  of  immunity  upon  the  animals.  As  has 
been  mentioned  before,  the  organism  of  cholera  may  be  injected 
subcutaneously  without  producing  disease,  and  within  certain  limi- 
tations aids  in  the  protection  against  invasion  by  these  organisms 
through  the  intestinal  canal. 


CHAPTER  II 

GENERAL  CONDITIONS  OF  INFECTION  AND 
RESISTANCE 

THE  PRODUCTION  OF  INFECTIONS. 

ENTRANCE  OF  THE  INVADING  ORGANISM. 

TYPES    OF    INFECTIOUS    DISEASES. 
FACTORS  FAVORING  THE  INVADER. 
FACTORS  INHIBITING  THE  INVADER. 

FACTORS  OPERATING  AGAINST  RESISTANCE  OF  HOST. 
FACTORS  FAVORING  THE  HOST. 
THE  COURSE  OF  ACUTE  INFECTIOUS  DISEASE. 

The  Production  of  Infection. — The  widespread  dissemination  of 
bacteria  in  nature  is  such  that  they  have  ready  access  to  plants  and 
animals.  Invasion  by  pathogenic  forms  may  set  up-  infection. 
Whether  or  not  the  infection  may  lead  to  disease  depends  upon  the 
final  relationship  established  between  the  invader  and  the  invaded 
body.  There  is  probably  no  condition  under  which  animals  or 
plants  fail  to  exhibit  some  degree  of  resistance  to  the  invading 
organism,  and  similarly  the  latter  attempts  to  accommodate  itself 
to  the  conditions  found  in  the  invaded  host.  If  the  resistance  be  not 
sufficient  to  overcome  the  invader,  infection  results.  The  produc- 
tion of  disease,  however,  depends  upon  the  superior  powers  of  the 
invader  over  the  resistance  of  the  host.  Occasionally  a  mutual 
adaptation  appears,  under  which  circumstances  an  animal  may  be 
infected  by  an  organism,  but  shows  no  symptom  or  sign  of  dis- 
ease. Not  infrequently  the  trypanosoma  Lewisi  is  found  in  the 
blood  stream  of  rats,  the  rats  continuing  to  live  an  apparently  nor- 
mal existence.  A  similar  mutual  adaptation  is  found  in  the  "  carrier 
state,"  wherein  man  may  harbor  virulent  diphtheria  bacilli  or  other 
organisms  without  any  evidence  of  disease.  Mutual  adaptation  is 
not  attained  without  a  struggle  on  the  part  of  both  invader  and 
host,  and  infectious  disease  results  when  the  invading  organism 
triumphs.  This  does  not  mean  permanence  of  infection,  because 
even  although  disease  is  established,  the  defenses  of  the  host 
continue  to  operate,  and  often  are  augmented  in  such  a  way  that 
ultimately  the  infection  disappears.  This  accounts  for  the  self- 
limitation  of  most  of  the  acute  infectious  diseases.  The  increase  in 
defensive  powers  may  in  certain  diseases  become  permanent  and 
immunity  thereby  be  established.  In  all  cases  of  recovery  from 
acute  infections  immunity  of  some  duration  appears,  although  it  may 
be  limited  to  a  few  weeks  or  a  few  months. 

Entrance  of  the  Invader. — The  entrance  of  the  invading  organ- 
ism may  be  due  to  an  interruption  of  continuity  of  those  surfaces  of 
the  body  which  ordinarily  are  impermeable  to  bacterial  invasion. 
These  surfaces  include  skin  and  the  mucous  membranes  of  the  re- 
spiratory, alimentary,  and  genito-urinary  tracts.  The  interruption 

ii 


12  THE  PRINCIPLES  OF  IMMUNOLOGY 

of  continuity  may  be  due  to  trauma  or  may  result  from  profuse 
growth  of  bacteria  on  the  surface  with  the  elaboration  of  poisonous 
products  which  may  kill  the  epithelial  cells.  The  former  condition 
is  exemplified  in  infected  wounds  and  the  latter  in  infection  by 
diphtheria  bacilli,  streptococci,  and  fungi,  such  as  produce  favus, 
thrush,  and  pityriasis.  Entrance  may  be  favored  by  changes  in  the 
character  of  secretions,  as,  for  example,  the  reduction  of  acidity  of 
the  gastric  juice  in  certain  forms  of  chronic  gastritis.  The  bacteria 
may  be  implanted  in  some  site  which  favors  their  multiplication,  as, 
for  example,  in  the  crypts  of  the  tonsils,  in  the  crevices  between 
unclean  teeth  and  in  hair  follicles.  Multiplication  in  these  situa- 
tions favors  the  production  of  poisonous  products  which  may  by 
destruction  of  cells  serve  to  interrupt  surface  continuity.  Somewhat 
similar  is  the  fact  that  extensive  destruction  of  tissues  may  provide 
dead  material  in  which  saprophytes  may  develop,  and  if  this  mate- 
rial is  so  deep  as  to  be  excluded  from  the  access  of  air,  conditions 
favorable  to  the  development  of  anaerobes  are  produced.  Infec- 
tion may  be  favored  by  the  movement  of  cells  and  fluids.  For 
example,  although  leucocytes  may  take  up  bacteria,  they  do  not 
invariably  destroy  them,  and  the  migration  of  such  leucocytes  may 
lead  to  the  dissemination  of  organisms  by  the  subsequent  death  of 
the  leucocyte.  The  movement  of  lymph  may  favor  invasion  as  is 
seen  not  uncommonly  in  those  cases  of  infections  of  the  hand  by 
streptococcus,  wherein  the  lymph  flow  carries  the  organisms  so  as 
to  set  up  infections  of  the  lymph-vessels  and  the  lymph-nodes,  and 
even  of  the  blood  stream.  Gaining  access  to  the  blood,  the  circula- 
tion of  this  fluid  tissue  may  deposit  bacteria  in  numerous  foci 
throughout  the  body.  The  route  of  invasion  depends  somewhat 
upon  the  type  of  organism,  those  of  typhoid  fever,  dysentery, 
and  cholera,  gaining  access  to  the  intestinal  canal  through  the 
mouth.  Their  implantation  upon  the  skin  is  of  no  significance, 
except  that  they  may  thence  be  transferred  to  the  mouth.  The 
gonococcus  produces  no  lesions  of  the  intestinal  canal,  but  implanted 
in  the  genital  tract,  the  eye,  or  the  endocardium  leads  to  serious 
results.  If  plague  bacilli  be  inoculated  subcutaneously  in  rats  a 
large  percentage  of  the  animals  survive,  but  if  implanted  in  the 
lower  respiratory  tract  small  doses  suffice  to  produce  fatal  infec- 
tions. The  pneumococcus  appears  to  infect  man  only  through  the 
respiratory  tract.  This  phenomenon  probably  depends  in  part  upon 
a  local  susceptibility  to  the  organisms. 

Types  of  Infectious  Disease. — The  types  of  infectious  disease 
are  differentiated  according  to  the  method  of  invasion  and  dissem- 
ination. An  organism  may  grow  locally  and  produce  only  local 
manifestations,  as  seen  in  a  small  abscess.  It  may  grow  locally  and 
produce  marked  general  disturbances,  as  is  the  case  in  diphtheria,  in 
which  instance,  although  organisms  may  enter  the  blood  stream, 
they  are  usually  confined  to  some  focus,  such  as  the  tonsils.  They 
elaborate  in  that  situation  poisonous  substances  which  are  absorbed 
and  set  up  general  manifestations  of  intoxication.  Certain  other 


INFECTION  AND  RESISTANCE  13 

diseases  may  produce  marked  local  manifestations  and  rapidly  in- 
vade the  blood  stream,  as  is  true  of  typhoid  fever.  This  organism 
enters  the  lymph-nodes  of  the  intestinal  tract,  produces  enlarge- 
ment, softening,  and  necrosis.  The  diarrhoea  in  these  cases  is  largely 
if  not  wholly  due  to  the  local  lesions,  but  the  severe  general  mani- 
festations are  due  principally  to  the  entrance  of  the  organisms  into 
the  blood  stream.  Other  diseases  may  show  little  local  manifesta- 
tion, as  is  true  of  tetanus,  but  even  with  slight  local  disturbances 
profound  general  symptoms  occur  as  the  result  of  absorption  of 
toxin.  Other  diseases,  such  as  anthrax,  may  show  little  local  mani- 
festation, but  rapidly  exhibit  generalized  infection  through  the  blood 
stream.  Infection  then  simply  signifies  successful  invasion.  Bac- 
teremia  signifies  the  presence  of  organisms  in  the  blood.  Septicemia 
signifies  blood  infection  associated  with  the  production  of  toxic 
substances.  Pyemia  indicates  that  bacteria  are  present  in  the  blood 
stream  and  because  of  lodgment  in  numerous  situations  produce 
multiple  abscesses.  Sapremia  indicates  absorption  of  toxic  products 
from  the  growth  of  saprophytic  organisms.  Primary  infections  are 
those  which  occur  without  any  decrease  of  resistance  due  to  another 
infection.  Secondary  infections  occur  in  individuals  already  suffer- 
ing from  an  infection  of  another  nature.  Such  an  infection  is  well 
exemplified  in  the  secondary  infection  of  a  tuberculous  cavity  of  the 
lung  by  staphylococcus.  Terminal  infections  are  those  which  occur 
near  the  fatal  termination  of  some  other  disease,  whether  that  other 
disease  be  of  bacterial  nature  or  of  some  other  origin.  Infections  of 
this  type  are  seen  in  the  terminal  broncho-pneumonias  and  sep- 
ticemias  which  occur  in  the  course  of  certain  chronic  diseases. 
Mixed  or  multiple  infections  are  not  rare  and  it  is  sometimes  diffi- 
cult to  determine  which  infection  is  of  greater  importance.  There  is  no 
doubt  that  one  infection  influences  another  existing  at  the  same  time  and 
usually  in  a  manner  deleterious  to  the  patient.  Infection  with  measles  or 
lobar  pneumonia  may  excite  latent  tuberculosis  into  activity.  Duke 
reports  the  lighting  up  of  latent  syphilis  by  an  attack  of  typhoid 
fever  and  of  latent  gonorrhea  by  an  attack  of  tonsillitis.  The  re- 
moval of  one  chronic  infection  may  favorably  influence  another,  as 
seen  in  the  relief  of  certain  cases  of  pyorrhea  alveolaris  by  the  re- 
moval of  infected  tonsils  and  in  numerous  other  instances  of  mul- 
tiple chronic  infections. 

Factors  Favoring  the  Invader. — The  small  size  of  pathogenic 
bacteria  and  protozoa  aids  in  their  avoidance  of  detection,  favors 
transportation,  and  aids  in  penetration.  The  rapidity  of  multiplica- 
tion of  such  organisms  is  of  considerable  importance  to  their  patho- 
genic powers.  Those  bacteria  which  form  spores  resist  destructive 
agents  and  can  resume  activity  when  favorable  conditions  present. 
Certain  of  the  protozoa,  more  particularly  the  endamebae,  are  cap- 
able of  forming  cysts  which  are  more  resistant  to  unfavorable  en- 
vironment than  the  active  organism.  Either  in  the  active  state  or 
in  the  vegetative  state,  organisms  may  persist  for  a  long  time  in  the 
so-called  carriers,  in  intermediate  hosts,  or  living  as  saprophytes. 


i4  THE  PRINCIPLES  OF  IMMUNOLOGY 

The  microparasites,  therefore,  can  be  said  to  have  a  ready  adapta- 
bility to  varying  environment  and  to  be  aided  in  propagation  by 
their  ability  to  derive  nutrition  from  food-stuffs  which  possess 
wide  differences  in  constitution.  Certain  bacteria  apparently  can 
produce  their  own  protein  from  amino-acids  and  have  no  diffi- 
culty in  deriving  nutrition  from  whole  proteins.  As  has  previ- 
ously been  indicated,  those  factors  which  go  to  increase  virulence 
of  organisms,  such  as  capsule  formation  and  the  production  of  toxic 
substances,  aid  materially  in  invasion. 

Factors  Inhibiting  the  Invader. — Although  rapid  multiplication 
aids  invasion,  nevertheless,  the  brief  life  period  which  most  of  the 
microparasites  exhibit  is  an  influence  operating  against  rather  than 
in  favor  of  invasion.  Many  pathogenic  organisms  are  susceptible 
to  the  destructive  influence  of  light,  heat,  desiccation,  etc.  In  cer- 
tain instances  the  life  of  organisms  outside  an  animal  body  operates 
to  reduce  virulence  and  therefore  to  inhibit  the  capacity  for  inva- 
sion and  production  of  disease. 

Factors  Operating  Against  Resistance. — The  animal  host  is  sub- 
jected to  the  attacks  of  invading  organisms  because  of  the  multi- 
plicity of  contacts  with  the  environment.  The  large  body  surface 
and  locomotion  of  the  body  are  influences  favoring  approximation 
of  the  invader.  Certain  living  activities,  such  as  the  ingestion  of 
foods  and  water,  coitus,  and  the  ready  availability  of  superficial 
orifices,  such  as  the  nose,  ears,  mouth,  anus,  genital  orifices,  all  aid 
invasion.  The  fact  that  most  animals  have  a  constant  body  tem- 
perature and  that  their  tissues  are  continually  moist,  provides  con- 
ditions favorable  to  the  invading  organism.  Although  light  rays 
beyond  the  violet  end  of  the  spectrum  have  a  certain  capacity  for 
the  penetration  of  tissues,  yet  ordinary  sunlight  exhibits  very  little 
penetrability;  therefore,  the  construction  of  the  body  is  such  that 
the  inhibitory  effect  of  light  is  not  brought  to  bear  upon  organisms 
that  have  already  gained  entrance.  The  anatomy  of  the  body  pro- 
vides certain  structures  which  are  relatively  inactive,  such  as  the 
appendix  vermiformis  and  the  crypts  of  the  tonsils  where  organ- 
isms find  moisture,  warmth,  and  darkness,  suitable  for  their  de- 
velopment. In  chronic  infections,  particularly  by  the  tubercle 
bacillus,  necrotic  tissues,  or  actual  cavities  may  exist  in  contact  with 
surfaces  and  with  the  outer  air,  and  both  conditions  operate  to  re- 
duce resistance  by  providing  favorable  places  for  bacterial  multipli- 
cation. The  circulation  of  lymph  and  blood  may  operate  against 
the  host  if  organisms  are  particularly  virulent.  Inspiration  of  con- 
taminated air  may  also  serve  to  aid  invaders.  The  resistance  of  the 
host  may,  in  a  manner  as  yet  unexplained,  be  decreased  in  general 
by  bodily  fatigue,  exposure  to  heat  and  cold,  poor  hygienic  sur- 
roundings, deleterious  gases,  or  improper  diet.  The  extremes  of 
life,  childhood  and  age,  are  associated  with  reduced  resistance.. 
Drugs,  operative  procedures,  improper  diet,  and  similar  conditions 
favor  infection. 

Factors  Favoring  the  Host.— The  possession  of  intelligence  by 


INFECTION  AND  RESISTANCE  15 

the  higher  forms  of  animal  life  aids  in  the  detection  and  elimination 
of  infective  organisms.  Not  only  may  this  be  accomplished  by  vol- 
untary movement,  but  the  purposeful  action  of  involuntary  re- 
flexes may  similarly  aid  the  host.  The  body  possesses  a  variety  of 
defenses  in  the  form  of  structure,  secretions,  chemical  substances, 
cellular  activity,  all  of  which  serve  to  aid  in  its  protection  in  connec- 
tion with  natural  and  acquired  resistance  to  disease.  These  will  be 
discussed  in  the  next  chapter.  Plants  produce  certain  diastases, 
aromatic  products,  aldehydes,  and  other  substances  which  create  in 
the  plant  a  state  deleterious  to  germination  of  harmful  invaders. 
Pigments  such  as  chlorophyl  may  destroy  toxic  substances  and 
even  bacteria  in  a  manner  somewhat  similar  to  the  action  of 
bile  pigment. 

The  Course  of  Infectious  Disease. — The  exact  moment  of  inva- 
sion of  an  infectious  agent  is  difficult  to  determine,  but  in  cases  of 
infectious  disease,  the  time  of  exposure  to  infection  can  usually  be 
stated  to  have  occurred  within  the  limits  of  a  few  hours.  Following 
the  moment  of  invasion  there  occurs  a  period  of  incubation  during 
which  the  host  exhibits  no  symptom  of  infection.  This  period  of 
incubation  in  some  diseases  is  extremely  variable,  whereas  in  others 
it  is  relatively  fixed.  In  diphtheria  incubation  may  apparently  vary 
from  twenty-four  hours  up  to  nine  or  ten  days,,  and  certain  other 
diseases  show  similar  variation.  In  scarlet  fever,  on  the  other  hand, 
the  incubation  period  is  very  commonly  five  days,  and  numerous 
other  diseases  show  similar  fixity  of  incubation  time.  Following 
the  period  of  incubation  the  less  violent  infectious  diseases  show  a 
short  period  of  prodromal  symptoms  in  which  headache,  malaise, 
and  other  minor  manifestations  may  appear.  The  next  period,  that 
of  onset  of  disease  or  so-called  invasion,  may  be  frank  or  insidious. 
Lobar  pneumonia  may  develop  within  a  period  of  a  few  hours  and 
exemplifies  frank  onset.  As  a  contrast,  typhoid  fever  is  likely  to 
occupy  a  week  or  ten  days  between  the  period  of  prodromal  symp- 
toms and  the  full  development  of  disease,  thus  illustrating  insidious 
onset.  That  period  during  which  the  disease  is  at  its  height  is  called 
the  fastigium  or  acme.  Following  the  fastigium  comes  the  period 
of  decline  or  defervescence.  This  may  be  by  crisis  or  lysis.  Crisis 
is  seen  in  approximately  half  the  cases  of  lobar  pneumonia,  in 
which  the  decline  occurs  in  a  period  of  a  few  hours.  Deferves- 
cence by  lysis  is  seen  in  a  large  number  of  infectious  diseases  and  is 
particularly  well  exemplified  by  typhoid  fever  in  which  several  days, 
a  week,  or  more,  may  be  consumed.  Convalescence  indicates  that 
period  during  which  the  symptoms  of  disease  have  practically  dis- 
appeared and  the  patient  gradually  recovers  and  is  restored  to  nor- 
mal. At  any  period  the  infection  may  become  so  overwhelming  as 
to  cause  the  death  of  the  individual.  Chronic  infectious  diseases 
exhibit  no  such  regularity  of  development  and  decline.  In  con- 
trast to  the  acute  infections,  these  are  not  likely  to  be  self-limited, 
but  progress  until  they  have  reached  a  point  of  such  great  severity, 
or  of  such  complete  exhaustion  of  the  host  that  death  ensues. 


CHAPTER  III 
THE  GENERAL  PHENOMENA  OF  IMMUNITY 

TYPES  OF  IMMUNITY. 

NATURAL  IMMUNITY. 
SPECIES. 
RACIAL. 
FAMILY. 
INDIVIDUAL. 
INHERITED   IMMUNITY. 
ACQUIRED  IMMUNITY. 

NATURALLY  ACQUIRED. 
ARTIFICIALLY  ACQUIRED. 

ACTIVE  ARTIFICIALLY  ACQUIRED. 

INOCULATION  OF  LIVING  VIRUS  IN   HEALTH. 
USE  OF  ATTENUATED  VIRUS. 
USE  OF  DEAD  BACTERIA. 
USE  OF   BACTERIAL   PRODUCTS. 
PASSIVE  ARTIFICIALLY   ACQUIRED. 
THEORIES  OF  THE  NATURE  OF  IMMUNITY. 
THE  EHRLICH  SIDE-CHAIN  THEORY. 
THE  EHRLICH  CLASSIFICATION  OF  IMMUNE  BODIES. 
CRITICISM   OF  THE  EHRLICH    HYPOTHESIS. 
THE   SPECIFICITY   OF   IMMUNE  REACTIONS. 

NON-SPECIFIC  THERAPY  OF  INFECTIOUS  DISEASES. 
THE  SITE  OF  ANTIBODY   FORMATION. 

PRODUCTION  OF  ANTIBODIES  AT  SITE  OF  INJECTION. 

Types  of  Immunity. — Resistance  to  disease  may  be  natural  or 
acquired.  If  natural  it  may  be  of  a  species,  race,  family,  or  indi- 
vidual character.  If  acquired  it  may  be  naturally  acquired,  as  seen 
in  the  immunity  following  an  attack  of  infectious,  disease,  or  it  may 
be  artificially  acquired.  If  artificially  acquired  it  may  be  the  result 
of  active  immunization  or  of  passive  immunization.  Artificially 
acquired  active  immunity  is  such  as  may  follow  the  injection  of 
various  antigens,  such  as  toxins,  bacteria,  and  numerous  other  sub- 
stances. Artificially  acquired  passive  immunity  is  the  result  of 
transfer  of  active  immunity  from  an  immune  animal  to  a  normal 
animal,  which  latter  becomes  passively  immunized. 

Natural  Immunity. — Although  the  term  immunity  may  be  consid- 
ered as  equivalent  to  the  capacity  for  resisting  disease,  nevertheless, 
in  common  usage  it  often  implies  an  increase  of  resistance.  In  esti- 
mating an  increase  of  resistance  a  normal  degree  must  be  presup- 
posed and  the  determination  of  the  normal  is  extremely  difficult. 
In  considering  natural  immunity  the  term  is  used  in  contrast  to  sus- 
ceptibility and  is  not  comparable  to  a  normal  level  of  resistance. 
Natural  resistance  to  disease  is  favored  by  structure,  movement, 
fluids,  and  secretions  of  the  body.  Structurally  the  skin  is  practi- 
cally impermeable  to  bacteria.  In  a  general  way  this  is  true  of 
mucous  membranes,  although  we  know  that  certain  organisms  may 
pass  through  mucous  membranes  of  the  intestinal  tract  without  any 
16 


GENERAL  PHENOMENA  OF  IMMUNITY  17 

lesions  of  the  surface.  Crypt-like  structures,  such  as  hair  follicles, 
sweat  glands,  crypts  of  the  tonsils,  gastro-intestinal  glands,  urethral 
glands,  may  serve  as  foci  where  bacteria  are  able  to  multiply,  and 
may  thus  determine  penetration  by  the  organism.  Accessory  struc- 
tures of  the  skin,  such  as  the  hairs  of  the  anterior  nares  and  the 
cilia  of  certain  parts  of  the  respiratory  tract,  aid  in  either  filtering 
the  air  or  in  propelling  lodged  organisms  toward  external  orifices. 
The  nature  of  certain  secretions  may  be  antagonistic  to  the  growth 
of  certain  bacteria  either  by  virtue  of  chemical  substances,  such  as 
the  hydrochloric  acid  of  the  gastric  juice,  normal  alkali  of  the  saliva 
and  upper  intestinal  tract,  or  by  virtue  of  digestive  ferments  which 
may  act  deleteriously  upon  bacterial  growth.  The  movement  of 
secretions,  as,  for  example,  that  of  the  conjunctival  sac,  may  favor 
the  elimination  of  organisms.  Bodily  movement  is  of  considerable 
value  in  resistance  to  infection,  whether  it  be  the  simple  process  of 
wiping  away  irritating  substances  or  the  more  intricate  process  of 
bathing  either  with  water  or  with  definite  anti-bacterial  fluids.  Re- 
flexes such  as  coughing,  sneezing,  and  vomiting  are  definitely  pur- 
posive in  protection.  The  movement  of  materials  in  the  intestinal 
canal  serves  to  prevent  any  too  great  bacterial  activity,  and  if  in 
spite  of  normal  intestinal  movement  irritative  substances  are  formed, 
the  response  by  diarrhea  serves  a  useful  purpose  in  elimination. 
Internally  the  fluids  of  the  body,  more  particularly  the  blood,  con- 
tain definite  anti-bacterial  and  anti-infective  substances.  In  addi- 
tion to  these  the  non-specific  ferments  of  the  body  fluids  aid  in 
combating  infection.  The  acidity  or  alkalinity  of  fluids  within  the 
body,  as  well  as  certain  substances  of  unknown  nature,  may  serve 
to  retard  or  prevent  bacterial  invasion.  Of  great  importance  in  pro- 
tection is  the  reaction  of  inflammation.  In  the  course  of  this  process 
fluids  and  cells  are  exuded  from  the  vessels.  The  exudation  of  fluids 
upon  surfaces  aids  in  washing  away  bacteria,  as,  for  example,  the 
profuse  exudation  of  fluid  in  acute  coryza  and  acute  enteritis.  Accu- 
mulation of  fluids  may  serve  to  dilute  bacterial  poisons  and  by  diffu- 
sion and  absorption  aid  in  the  elimination  of  these  poisons.  The 
cells  which  form  part  of  the  exudate  possess,  as  characteristic  func- 
tions, the  capacity  of  taking  up  bacteria  by  phagocytosis  and  de- 
stroying them.  The  formation  of  fibrin  in  the  exudate,  as  well  as 
the  subsequent  proliferation  of  fixed  tissue  cells,  serves  to  delimit 
the  process  and  thereby  aid  in  the  prevention  of  widespread  dis- 
semination of  the  organisms.  In  superficial  inflammations  the  ex- 
foliation of  diseased  cells,  as  in  scarlatina,  may  aid  in  the  elimination 
of  the  infective  virus.  This  does  not  mean,  however,  that  such  cells 
retain  an  infective  character  after  long  periods  of  desiccation. 

The  physiological  activity  of  cells  in  the  body  may  be  so  excited 
as  to  aid  in  the  elimination  of  toxic  products,  as  exemplified  by  the 
early  increase  of  activity  in  infectious  disease.  If  the  toxic  mate- 
rial be  sufficiently  virulent  this  period  of  hyperactivity  may  be  sue- 


i8  THE  PRINCIPLES  OF  IMMUNOLOGY 

ceeded  by  one  of  depression.  The  stimulation  of  cells  in  the  produc- 
tion of  antitoxic  and  anti-bacterial  substances  will  be  discussed  sub- 
sequently. 

Classification  of  Natural  Immunity — Species  Immunity. — As  has 
been  indicated  above,  natural  immunity  may  be  found  in  species, 
races,  families,  or  individuals.  It  is  profitable  to  emphasize  again 
that  what  we  speak  of  as  species  immunity  expresses  a  difference  in  sus- 
ceptibility exhibited  by  certain  species  as  contrasted  with  others. 
Whereas  man  is  susceptible  to  such  diseases  as  syphilis,  gonorrhea, 
cholera,  and  diphtheria,  numerous  other  species  are  resistant  to  these 
diseases.  It  is  possible  to  inoculate  syphilis  in  higher  apes,  in  the 
rabbit,  possibly  in  the  guinea-pig  and  other  animals,  but  even  suc- 
cessful inoculation  shows  a  greater  degree  of  resistance  than  is  pos- 
sessed by  man.  Conversely,  man  is  not  susceptible  to  hog-cholera, 
chicken-cholera,  rat-typhoid,  and  certain  other  diseases.  Man  is 
susceptible  to  the  bacillus  of  human  tuberculosis,  but  less  so  to 
that  of  bovine  tuberculosis,  still  less  to  that  of  avian  tuberculosis, 
and  not  at  all  to  that  of  fish  tuberculosis.  In  fact,  with  the  exception 
of  the  rabbit,  fish  tuberculosis  is  not  transferable  to  any  of  the  warm- 
blooded animals.  Fish  are  not  susceptible  to  human  tuberculosis. 
Practically  all  animals  are  susceptible  to  snake  venoms  except  the 
hog.  Man  is  highly  susceptible  to  pneumococcus  and  to  bacillus 
pestis,  but  fowl  are  resistant  to  both  these  organisms.  Metchnikoff 
showed  that  certain  species  of  insects  are  susceptible  to  diphtheria 
toxin  whilst  others  are  not.  Man  is  susceptible  to  trypanosoma  gam- 
biense,  but  is  resistant  to  trypanosoma  naganae.  In  some  instances 
these  variations  in  susceptibility  and  resistance  depend  upon  the 
environment.  For  example,  frogs  kept  in  low  temperature  are  not 
susceptible  to  anthrax,  but  if  kept  in  a  temperature  of  35°  C.  they 
succumb  to  the  disease.  Similarly  it  was  found  that  if  lizards  are 
kept  at  16°  C.  they  could  not  be  infected  with  plague,  but  at  a  higher 
temperature  were  susceptible.  The  work  of  Pasteur  with  anthrax 
in  fowl  is  a  classical  experiment.  He  found  that  if  he  kept  fowl  at 
low  temperatures  they  became  susceptible  to  anthrax  because  of  the 
decrease  of  body  temperature ;  but  if  they  were  allowed  to  maintain 
their  normally  high  body  temperature  they  were  resistant.  The 
temperature  of  most  of  the  lower  mammalia  is  higher  than  that  of 
man,  but  the  difference  is  not  sufficiently  great  to  explain  all  the  varia- 
tions in  susceptibility  and  resistance. 

Racial  immunity  probably  exists  but  cannot  be  so  conclusively 
proven  in  man  as  is  true  of  species  immunity.  It  is  generally  be- 
lieved that  Caucasians  are  less  susceptible  to  tuberculosis  than 
negroes.  That  this  is  an  inherent  character  of  the  race  appears  to  be 
somewhat  doubtful.  Difference  in  hygienic  conditions  and  in  de- 
gree of  exposure  to  the  disease  may  account  for  much  that  appears 
to  be  racial  susceptibility.  It  is  possible  that  the  superior  hygienic 
conditions  of  whites  in  northern  latitudes  explains  this  difference. 
It  is  also  possible  that  having  been  the  victims  of  tuberculosis  for 


GENERAL  PHENOMENA  OF  IMMUNITY  19 

many  centuries  a  certain  degree  of  racial  immunity  has  been  estab- 
lished by  virtue  of  the  elimination  of  more  susceptible  individuals 
and  the  survival  of  the  more  resistant.  It  is  apparently  true  that 
when  an  infectious  disease  first  attacks  a  race,  it  is  more  virulent 
than  in  those  races  where  it  is  commonly  found.  The  native  African 
when  brought  into  contact  with  tuberculosis  appears  to  be  attacked 
violently.  The  decimation  of  the  population  of  Iceland  after  the 
introduction  of  measles  was  one  of  the  horrors  of  improved  com- 
munications ;  subsequent  epidemics  of  the  disease  in  the  same  people 
have  been  considerably  less  fatal.  The  introduction  of  syphilis  into 
the  American  Indian  showed  a  virulence  unknown  among  the  Cau- 
casians. Smallpox  materially  aided  the  Spaniard  in  his  conquest  of 
Mexico.  The  negro  is  supposed  to  be  less  susceptible  to  yellow 
fever  than  is  the  Caucasian,  but  careful  investigation  would  make 
it  appear  that  in  infancy  and  childhood  acquired  immunity  is  estab- 
lished by  mild  attacks  of  the  disease.  The  recent  work  of  Love  and 
Davenport  shows  that  among  500,000  troops  illness  was  19  per  cent, 
more  frequent  among  negro  than  among  white  troops.  The  negro 
was  apparently  less  resistant  to  pneumonia,  tuberculosis,  and  small- 
pox than  the  white.  The  negro  was  more  resistant  to  skin  diseases, 
but  contracted  venereal  disease  readily  and  suffered  more  than  the 
whites  from  extension  and  complications  of  venereal  disease.  Borell 
has  reported  that  the  Senegalese  are  very  susceptible  to  pneumonia 
even  in  their  own  country.  On  transportation  to  France  during  the 
World  War  more  than  5  per  cent,  succumbed  to  pneumonia  before 
they  had  become  acclimated,  but  in  those  who  had  been  in  France 
two  or  three  years,  the  death-rate  from  pneumonia  was  much  re- 
duced ;  only  2  in  7000  troops  died  of  pneumonia.  Whether  this 
reduction  is  due  to  acclimatization  or  the  early  elimination  of  the 
more  susceptible  is  an  open  question.  An  apparent  racial  immunity 
to  malaria  may  be  explained  by  the  persistence  of  this  disease  for 
many  years  following  a  childhood  infection.  In  Australia,  New 
Zealand,  and  Tasmania  during  the  years  1906-1908  there  were  only 
about  half  the  deaths  per  thousand  inhabitants  as  the  result  of  tuber- 
culosis than  occurred  in  Ireland,  Norway,  and  Japan,  during  the  same 
period ;  whilst  the  rate  decreased  regularly  in  the  former  countries 
it  increased  in  the  latter.  This  appears  to  favor  the  idea  of  racial 
differences  of  susceptibility,  but  a  careful  analysis  of  all  the  condi- 
tions may  show  that  climate,  mode  of  life,  and  hygienic  conditions 
have  a  considerable  influence.  In  the  lower  animals  racial  differ- 
ence may  be  more  satisfactorily  illustrated.  Common  sheep  are  sus- 
ceptible to  anthrax,  whereas  the  Algerian  sheep  seem  to  be  immune. 
The  culex  mosquito  rarely  harbors  the  malarial  parasite,  whereas 
the  anopheles  are  commonly  infected.  The  field  mouse  is  highly 
susceptible  to  glanders,  whilst  the  white  mouse  is  immune.  The 
gray  mouse  is  more  resistant  to  streptococcus  infections  than  is  the 
white  mouse.  The  common  rat  is  more  resistant  to  anthrax  than 
is  the  white  rat. 


20  THE  PRINCIPLES  OF  IMMUNOLOGY 

Family  Immunity. — Members  of  certain  families  may  through 
generations  appear  to  be  especially  susceptible  to  such  diseases  as 
tuberculosis  and  rheumatism  or  the  converse  may  be  true.  In  the 
case  of  tuberculosis  this  difference  may  be  the  result  of  conforma- 
tion of  the  body.  The  physical  character  of  flat,  narrow  chest  and 
thin  skin  apparently  go  hand  in  hand  with  susceptibility  to  tuber- 
culosis, whereas  the  well-rounded  chest  appears  to  indicate  resist- 
ance. In  a  family  with  whose  history  we  are  familiar  the  blondes 
have  almost  invariably  succumbed  to  tuberculosis  and  the  brunettes 
living  under  the  same  conditions  and  in  intimate  association  have  been 
resistant.  This  must  be  due  to  inherent  constitutional  characters  and  is 
not  to  be  considered  as  a  difference  due  to  complexion  alone. 

Individual  Immunity. — Variations  of  individual  resistance  or  im- 
munity are  seen  frequently.  It  is  true  that  the  extremes  of  age 
show  a  certain  proneness  to  infection  and  that  this  varies  somewhat 
with  individuals.  Excellent  examples  of  individual  resistance  are 
seen  in  great  epidemics  where  some  of  those  exposed  apparently  in 
the  same  manner  and  under  the  same  hygienic  conditions  as  others 
show  either  complete  resistance  to  the  disease,  or,  if  they  are  at- 
tacked, develop  only  moderate  or  slight  attacks.  Infected  water  and 
foods  consumed  by  a  population  may  lead  to  disease  in  only  a  small 
portion  of  those  exposed.  Individual  variations  in  animals  are  very 
frequent  and  offer  a  considerable  source  of  error  in  the  interpreta- 
tion of  experimental  results.  If  a  series  of  guinea-pigs  be  injected 
with  the  same  dose  of  anthrax  bacilli,  all  will  die  at  practically  the 
same  time,  but  if  rabbits  be  treated  in  the  same  way  some  die 
within  two  days,  others  die  subsequently,  and  still  others  are  com- 
pletely resistant.  On  the  other  hand,  rabbits  are  all  susceptible  to 
chicken-cholera,  whereas  the  guinea-pig  shows  great  individual  dif- 
ference. Although  a  large  number  of  children  suffer  from  tonsillar 
infections,  yet  the  incidence  of  acute  articular  rheumatism  or  of 
endocarditis  is  small  and  variable.  Instances  might  be  multiplied 
indefinitely  of  individual  variations  in  resistance,  but  the  phenom- 
enon is  one  of  common  knowledge. 

Inherited  Immunity. — The  immunity  transferred  from  parent  to 
offspring  may  be  a  natural  immunity  or  an  immunity  acquired  by 
the  parent.  The  transfer  of  natural  immunity  may  be  seen  in  racial, 
species,  and  family  manifestations,  and  is  probably  a  true  transfer 
through  the  germ  plasm.  Congenital  immunity  may  arise  either  in 
the  form  of  an  active  immunity  developed  in  the  fetus  because  of 
the  presence  of  antigens  in  the  circulating  blood  of  the  mother,  or 
may  be  in  the  form  of  passive  immunity  transferred  from  the  blood 
of  the  mother  to  that  of  the  fetus.  It  is  conceivable  that  the  fetus 
may  survive  an  attack  of  disease  transmitted  from  the  mother  and 
thereby  become  immune.  It  has  been  known  since  the  time  of 
Pasteur  that  certain  dogs  are  immune  to  rabies.  Remlinger  has 
found  that  the  guinea-pig  may  transfer  rabies  to  the  fetus  and 
puppies  have  been  known  to  become  rabid  several  months  after 


GENERAL  PHENOMENA  OF  IMMUNITY  21 

birth  without  any  evidence  of  having  been  bitten,  the  disease  there- 
fore probably  having  been  contracted  in  utero.  Immunity  in  dogs 
may  be  explained  by  direct  transmission  of  immunity  from  the 
mother,  or  by  survival  of  the  disease  in  uterine  or  early  post- 
uterine  life. 

Acquired  Immunity— ^Naturally  Acquired  Immunity. — The  acqui- 
sition of  immunity  may  be  through  so-called  natural  processes,  such 
as  passing  through  and  recovering  from  an  infectious  disease,  or  it 
may  be  induced  and  artificially  acquired  by  special  methods  of  im- 
munization to  be  described.  In  both  these  instances,  although  the 
normal  level  of  resistance  cannot  always  be  accurately  determined, 
yet  there  is  no  doubt  that  the  acquired  immunity  represents  a  higher 
level  of  resistance  than  is  normally  possessed.  For  example,  the 
fact  that  when  a  patient  has  survived  an  attack  of  such  a  disease  as 
scarlatina  and  then  in  spite  of  repeated  and  intimate  exposure  re- 
sists infection,  leaves  no  doubt  that  his  acquired  immunity  repre- 
sents a  higher  level  of  resistance  than  he  possessed  before  the  attack 
of  the  disease.  The  diseases  which  confer  a  lasting  immunity  include 
acute  anterior  poliomyelitis,  chickenpox,  cholera,  epidemic  cerebro- 
spinal  meningitis,  measles,  mumps,  plague,  scarlatina,  smallpox, 
typhoid  fever,  typhus  fever,  whooping-cough,  and  yellow  fever. 
The  question  as  to  whether  or  not  syphilis  confers  a  lasting  im- 
munity has  been  reopened  by  the  discovery  of  the  Wassermann  test 
and  by  the  work  of  Warthin.  The  Wassermann  test  has  shown  that 
many  cases  of  apparently  cured  syphilis  are  really  in  a  latent  stage 
of  the  disease.  Warthin  has  found  the  treponema  pallidum  in 
various  organs  at  autopsy  on  syphilitics  who  clinically  appeared  to 
be  free  from  the  disease.  If  Warthin's  work  can  be  confirmed  in  a 
large  number  of  cases  it  would  appear  that  syphilitic  infection  re- 
mains latent  throughout  the  life  of  the  individual  in  the  vast  ma- 
jority of  cases,  even  in  spite  of  the  fact  that  the  Wassermann  test  is 
negative  and  no  clinical  signs  of  the  disease  are  demonstrable.  If 
syphilis  be  curable,  the  reported  occurrence  of  second  infections  in  a 
small  number  of  instances  would  make  it  appear  that  any  immunity 
which  may  develop  is  not  permanent.  The  long  duration  of  the 
disease  would  account  for  the  small  number  of  reinfections  reported. 
Immunity  in  tuberculosis  has  been  extensively  studied,  and  as  yet 
no  final  and  conclusive  statements  can  be  made.  It  seems  probable 
that  tuberculosis  is  never  completely  eliminated  from  the  body,  and 
although  the  patient  exhibits  no  symptom  nor  sign,  he  still  may 
harbor  the  disease.  The  studies  of  Opie  and  others  would  make  it 
appear  that  the  development  of  tuberculosis  in  adult  life  is  traceable 
directly  to  old  lesions  which  occurred  in  childhood.  The  fact  that  a 
very  large  number  of  individuals  show  at  autopsy  small  lesions  indi- 
cates the  prevalence  of  the  disease.  Subsequent  active  development, 
following  encapsulation  of  a  lesion,  appears  to  be  due  to  certain  fac- 
tors which  either  reduce  the  protective  properties  of  the  body  or 
excite  the  organisms  to  renewed  activity,  or  both. 


22  THE  PRINCIPLES  OF  IMMUNOLOGY 

Artificially  Acquired  Immunity. — The  artificial  acquisition  of  im- 
munity may  be  the  result  of  active  development  of  immune  sub- 
stances in  the  organism  or  it  may  be  due  to  the  transfer  into  the 
organism  of  immune  substances  from  an  immune  animal.  Arti- 
ficially acquired  immunity  differs  from  naturally  acquired  immunity 
in  that  it  is  likely  to  be  less  durable.  If  acquired  by  active  immuniza- 
tion the  duration  is  likely  to  be  considerably  greater  than  if  acquired 
by  passive  immunization.  In  the  discussion  of  immunity  it  is  well 
to  keep  clearly  in  mind  the  definition  of  antigen  and  antibody.  The 
antigen  is  a  substance  which  upon  introduction  into  the  body  in 
proper  amounts  and  under  suitable  conditions  induces  the  formation 
of  a  special  antagonistic  substance,  the  antibody.  Conversely  the 
antibody  is  the  substance  produced  as  a  result  of  the  introduction 
of  antigen.  Experimentally  the  antigen  is  usually  introduced  by 
parenteral  routes,  meaning  routes  other  than  by  way  of  the  ali- 
mentary canal,  such  as  intravenous,  intraperitoneal,  subcutaneous, 
intrathecal,  intraocular,  and  by  other  similar  pathways.  The  nature 
of  antigens  and  antibodies  will  be  discussed  in  the  subsequent  chap- 
ters, but  it  may  be  said  here  that  both  are  of  protein  nature.  Every 
soluble  complete  protein,  with  the  exception  of  the  racemized  pro- 
tein of  Dakin,  may  serve  in  at  least  some  degree  as  an  antigen. 
The  proteins  employed  are  for  the  most  part  native,  but  synthetic 
proteins  may  also  act  as  antigens.  Wells  states  that  "  of  the  cleav- 
age products  of  proteins  it  is  certain  that  none  of  the  ammo-acids 
and  simple  polypeptids  can  act  as  antigens,  and  it  is  not  yet  fully 
established  that  even  such  large  complexes  as  the  proteoses  are 
antigenic,  although  there  is  some  evidence  in  favor  of  this  view." 
There  have  been  numerous  reports  of  the  use  of  lipoids  as  antigens, 
but  this  relation  has  not  been  definitely  established.  If  lipoids  are 
obtained  from  animal  tissues  favorable  results  may  be  obtained,  but 
in  none  of  these  experiments  is  it  proven  that  the  lipoids  are  entirely 
free  from  proteins.  Ford  has  successfully  employed  a  hemolytic 
glucoside  obtained  from  the  poisonous  mushroom  amanita  phalloides 
as  an  antigen  for  the  production  of  an  anti-hemolysin,  but  this  is 
the  only  well-established  exception  to  the  general  rule  that  antigens 
are  of  protein  nature. 

Actively  Acquired  Immunity. — This  may  be  produced  by  actual 
infection  of  an  individual  during  a  period  of  good  health  by  the 
virus  of  the  disease  to  which  he  is  to  be  immunized.  The  classical 
example  of  this  form  of  immunization  was  the  practice  for  many 
centuries  of  inoculating  smallpox  into  the  healthy,  so  as  to  induce 
a  mild  attack  of  the  disease.  The  danger  lies  in  the  uncertainty 
of  action  of  the  virus,  since  apparent  health  does  not  necessarily 
presuppose  resistance  to  any  special  disease.  If  the  virus  can  be 
measured  in  some  way  so  that  an  extremely  small  amount  can  be 
inoculated,  the  procedure  is  somewhat  safer.  Protection  against  Texas 
fever  in  cattle  has  been  practised  by  permitting  nursing  calves  to  be 


GENERAL  PHENOMENA  OF  IMMUNITY  23 

bitten  by  a  small  number  of  infected  ticks  or  by  injecting  intraven- 
ously a  small  amount  of  blood  from  an  infected  animal. 

Somewhat  similar  to  the  above  examples  is  infection  with  attenu- 
ated virus.  Such  attenuation  may  be  obtained  by  prolonged  cultiva- 
tion on  artificial  media,  by  heat,  by  passage  through  animals,  by 
desiccation,  by  the  use  of  chemical  agents,  and  by  pressure.  If  heat 
be  employed  for  attenuation,  rather  than  for  killing  the  organisms, 
it  must  be  properly  adjusted.  Toussaint  employed  this  method  in 
his  early  experiments  with  anthrax  in  which  he  heated  infected 
blood  to  55°  C.  for  ten  minutes.  This  method,  however,  is  not  re- 
liable, probably  because  of  variations  in  the  resistance  of  individual 
members  of  a  culture  of  any  given  organism.  Heat  may  be  applied 
also  during  the  cultivation  of  organisms  upon  artificial  media,  a 
method  practised  by  Pasteur  in  producing  anthrax  vaccine.  The 
heat  must  be  of  such  a  degree  as  to  permit  growth  of  the  organisms, 
but  at  the  same  time  reduce  the  virulence.  As  has  been  pointed 
out  before,  the  cultivation  of  organisms  upon  artificial  media  through 
many  generations  leads  to  a  reduction  of  virulence.  This  latter 
method  was  employed  by  Pasteur  in  the  development  of  the  vaccine 
for  chicken-cholera.  The  attenuation  of  smallpox  virus  by  passage 
through  the  calf  so  reduces  virulence  that  the  virus  may  safely  be 
inoculated  into  man.  Pasteur  found  that  the  virus  of  swine  ery- 
sipelas could  be  attenuated  by  passage  through  rabbits,  and  it  is 
well  known  that  the  passage  of  rabies  virus  through  dogs  and 
through  monkeys  reduces  its  virulence.  An  excellent  example  of 
attenuation  by  desiccation  is  found  in  the  preparation  of  anti-rabic 
vaccine.  For  this  purpose  the  virus  is  raised  by  passage  through 
rabbits  to  a  standard  degree  of  virulence,  the  "  virus  fixe."  The 
spinal  cord  of  a  rabbit  so  infected  is  desiccated  at  25°  C.  over  KOH. 
This  method  of  attenuation  is  so  delicate  that  there  are  distinct 
variations  in  virulence  between  fragments  dried  for  five,  six,  and 
seven  days,  as  well  as  virus  dried  for  thirty-five,  thirty-six,  and 
thirty-seven  days  or  intervening  periods.  The  longer  the  desicca- 
tion the  greater  the  reduction  of  virulence  and  the  greater  the  safety 
of  the  inoculation.  Attenuation  by  the  use  of  chemicals,  such  as 
phenol,  potassium  bichromate,  and  sulphuric  acid,  has  been  prac- 
tised. Chemical  attenuation  may  also  be  applied  to  toxins,  as  in  the 
use  of  iodine  terchloride  and  potassium  iodide.  A  pressure  of  eight 
atmospheres  at  a  temperature  of  28°  to  39°  C.  has  been  employed 
for  the  attenuation  of  anthrax  cultures,  but  is  probably  not  widely 
applicable,  is  difficult,  and  possesses  no  superior  advantages. 

Immunization  with  Dead  Bacteria. — In  the  study  of  immune 
processes  it  was  finally  found  that  killed  bacteria  could  be  used  for  the 
production  of  immunity.  The  organisms  may  be  killed  by  heat  or 
by  chemicals.  In  either  case,  it  is  necessary  so  to  apply  these  agents 
as  to  kill  the  organisms  without  destroying  their  proteins.  The 
use  of  heat  sufficiently  high  to  destroy  spores  leads  to  destruction 
also  of  the  proteins,  and  therefore  the  method  does  not  apply  to 


24  THE  PRINCIPLES  OF  IMMUNOLOGY 

spore^bearing  organisms.  Those  organisms  which  do  not  produce 
spores  can  be  killed  by  heat  of  58°  to  60°  C.  for  thirty  to  sixty 
minutes,  and  this  degree  of  heat  does  not  alter  the  character  of  the 
proteins.  'The  chemicals  most  frequently  employed  for  killing  bac- 
Iteria  so  as  not  to  alter  the  proteins  are  formaldehyde  and  phenol. 

Immunization  with  Bacterial  Products. — In  addition  to  the  use 
of  dead  bacteria,  as  indicated  above,  it  has  been  found  possible  to 
produce  immune  reactions  by  the  use  of  extracts  of  the  organisms, 
these  extracts  containing  a  considerable  amount  of  bacterial  pro- 
tein. Immunization  of  this  sort  leads  to  the  formation  of  anti- 
bacterial sera  which  agglutinate  the  bacteria  or  precipitate  bacterial 
extracts.  It  is  possible  also  that  this  method  of  immunization  leads 
to  the  formation  of  other  immune  substances.  How  far  protein, 
either  in  solution  or  in  the  bodies  of  bacteria,  may  be  broken  down 
and  still  be  capable  of  leading  to  the  formation  of  immune  bodies  is  a 
iquestion  that  has  been  extensively  studied.  Certainly  any  change 
that  breaks  up  the  protein  into  its  fundamental  amino-acids  is  likely 
to  destroy  its  antigenic  properties.  Simple  fractionation  by  means 
of  salting  still  leaves  sufficient  native  protein  to  serve  to  immunize. 

Of  bacterial  products  which  have  been  employed  for  immuniza- 
tion none  is  more  important  than  those  poisonous  bodies  called 
toxins.  In  the  classification  of  toxins  we  have  referred  to  the  true 
toxins  or  exotoxins  and  to  the  endotoxins.  There  is  little  support 
for  the  belief  that  endotoxins  as  such,  except  in  rare  instances,  can 
produce  immune  substances.  On  the  other  hand,  the  production  of 
a  neutralizing  antitoxin  against  the  exotoxins  has  constituted  one 
of  the  most  brilliant  chapters  in  the  study  of  immunology,  and  it 
will  be  given  discussion  in  the  chapter  on  toxins  and  antitoxins. 
The  use  of  toxins  as  antigens  involves  the  employment  of  these 
substances  in  non-fatal  doses,  their  attenuation  by  chemical  and 
physical  means,  or  their  primary  neutralization  by  means  of  previ- 
ously prepared  antitoxins.  In  experimental  work  on  animals  the 
first  two  methods  are  commonly  employed  and  may  be  combined 
with  the  third  method.  In  man  immunization  by  the  use  of  toxins 
is  practised,  mainly  in  connection  with  active  immunization  to  diph- 
theria. The  combination  between  toxin  and  antitoxin  is  not  in  the 
nature  of  a  fixed  and  final  reaction,  and  under  certain  circumstances 
partial  dissociation  may  occur.  The  active  immunization  of  man 
by  the  use  of  neutralized  mixtures  of  toxin  and  antitoxin  appears 
to  provide  conditions  whereby  dissociation  progresses  gradually, 
and  the  toxin  is  liberated  in  such  small  amounts  that  it  does  no 
harm  and  yet  induces  in  the  body  antitoxin  formation.  In  the  mean- 
time the  individual  is  protected  by  the  antitoxin  simultaneously 
dissociated.  Recent  studies  make  it  appear  that  several  organisms 
which  formerly  were  supposed  to  produce  only  endotoxins  elab- 
orate in  addition  true  toxins,  and  some  of  the  earlier  studies  sup- 
porting the  assumption  that  antitoxins  could  be  produced  by  these 


GENERAL  PHENOMENA  OF  IMMUNITY  25 

endotoxins  are  probably  fallacious,  because  of  the  mixture  of  un- 
recognized exotoxins,  the  latter  producing  the  immune  reaction. 

Active  immunization  may  be  produced  not  only  by  toxic  sub- 
stances elaborated  by  bacteria,  but  also  by  toxic  substances  produced 
in  animal  life,  such  as  snake  venoms,  spider  poisons,  and  similar 
substances.  Higher  plant  poisons,  such  as  ricin,  abrin,  crotin,  etc., 
may  produce  specific  neutralizing  antibodies.  The  practical  value 
of  the  antitoxins  prepared  against  bacterial  toxins  and  against  the 
venoms  produced  by  animals  is  such  as  to  have  added  greatly  to  the 
combating  of  poisoning  by  these  substances. 

Passive  Immunization. — In  active  immunization  the  animal 
manufactures  within  its  own  body  immune  substances  which  serve 
to  protect  against  and  combat  infection.  Passive  immunization, 
however,  utilizes  these  immune  substances,  through  the  transfer  of 
blood  serum  containing  the  products  of  active  immunization.  The 
most  common  example  of  passive  immunization  is  found  in  the 
therapeutic  use  of  diphtheria  antitoxin.  For  practical  purposes  the 
diphtheria  antitoxin  is  manufactured  in  the  body  of  the  horse.  The 
injection  of  immune  horse  serum  transfers  to  man  the  immunity 
actively  produced  in  the  horse.  Passive  immunity  of  this  sort  serves 
to  protect  against  infection,  and  until  the  possibility  of  active  im- 
munization of  man  against  diphtheria  was  demonstrated,  the  former 
method  was  widely  employed  for  protection  of  exposed  individuals 
against  diphtheria.  This  method  of  protection  has  the  great  advan- 
tage of  quickly  conferring  immunity  and  is  widely  employed  when 
time  does  not  permit  the  use  of  methods  for  developing  active  im- 
munity. After  the  disease  has  developed  the  use  of  immune  serum 
to  combat  the  infection  has  the  utmost  value.  In  the  case  of  tetanus 
antitoxin  the  protective  value  of  prophylactic  injections  has  been 
amply  demonstrated,  but  in  this  instance  the  great  affinity  between 
nerve  tissues  and  tetanus  antitoxin  is  such  that  the  therapeutic  use 
of  tetanus  antitoxin  after  the  disease  has  developed  has  not  given 
such  beautiful  results  as  has  been  true  of  the  serum  treatment  of 
diphtheria.  Much  encouragement  has  recently  been  afforded  by  the 
use  of  similarly  prepared  antitoxins  against  the  toxin  of  the  bacillus 
of  gas-gangrene,  and  there  is  little  doubt  that  the  methods  may  be 
much  more  widely  employed  as  it  becomes  possible  to  demonstrate 
the  formation  of  true  exotoxins  by  other  bacteria.  Not  only  may 
advantage  be  taken  of  substances  produced  by  artifically  acquired 
immunity,  but  in  certain  instances  it  is  feasible  to  use  the  blood 
serum  of  individuals  who  have  acquired  immunity  by  survival  of  an 
attack  of  certain  diseases.  In  this  field,  however,  the  facts  have  not 
been  accumulated  in  sufficient  number  to  justify  unqualified  ap- 
proval of  the  method. 

Passive  immunity  may  be  not  only  antitoxic  in  character,  but 
also  anti-bacterial.  Anti-bacterial  immune  sera  have  been  prepared 
against  the  streptococcus,  the  meningococcus,  the  pneumococcus, 
and  other  organisms.  The  success  with  passive  immunization  by 


26  THE  PRINCIPLES  OF  IMMUNOLOGY 

the  use  of  these  sera  has  not  always  been  so  clear  cut  as  in  the  case 
of  antitoxic  sera.  Nevertheless  the  use  of  anti-meningococcus  sera 
has  reduced  the  mortality  of  epidemic  cerebrospinal  meningitis  from 
75  or  80  per  cent,  down  to  35  or  40  per  cent,  or  lower.  The  results 
with  anti-streptococcus  sera  have  been  variable.  Although  the 
early  reports  of  the  use  of  anti-pneumococcus  sera  were  highly  en- 
couraging, later  study  has  thrown  some  doubt  upon  the  value  of 
this  method  of  treatment.  Much  further  study  of  the  subject  is  re- 
quired before  a  definite  conclusion  can  be  reached. 

Theories  of  the  Nature  of  Immunity. — In  the  early  study  of  im- 
munity numerous  hypotheses  were  advanced  as  to  the  action  and 
development  of  immune  bodies.  It  was  known,  for  example,  that 
when  bacteria  are  grown  for  a  long  time  upon  a  culture  medium 
certain  substances  are  produced  which  have  a  deleterious  influence 
upon  the  further  growth  of  the  organisms  and  may  actually  lead  to 
their  death.  It  was  easy  to  assume,  therefore,  that  recovery  from 
an  infectious  disease  might  be  due  to  the  development  of  similar 
antagonistic  substances  within  the  infected  host.  Another  theory 
was  to  the  effect  that  bacteria  growing  in  the  body  utilize  and 
exhaust  the  specific  nutritive  substances  necessary  for  their  growth 
and  then  die.  It  was  also  thought  that  the  death  of  bacteria  in  the 
body  was  due  to  changes  in  reaction  of  the  blood,  and  further  that 
altered  osmotic  conditions  changed  the  permeability  of  cell  mem- 
branes so  as  to  permit  ready  entrance  of  poisonous  substances. 
These  theories,  however,  could  not  withstand  the  demonstration  of 
passive  transfer  of  immunity,  the  production  of  immunity  by  the 
use  of  killed  organisms,  or  perhaps  more  important,  the  clear  demonstra- 
tion of  immune  reactions  in  vitro. 

The  Ehrlich  Side-chain  Theory. — As  more  and  more  facts  were 
added  to  the  knowledge  of  the  subject,  Ehrlich  propounded  his  side- 
chain  theory.  This  was  based  upon  the  law  of  Weigert,  which 
states  that  when  animal  cells  are  required  to  repair  an  injury 
they  not  infrequently  exceed  the  absolute  necessity  for  repair  and 
produce  tissue  in  excess.  Ehrlich,  therefore,  hypothesized  that  the 
injurious  substances  of  infection  demand  of  the  cells  the  forma- 
tion of  protective  bodies,  and  that  the  cells  respond  to  this  demand 
in  such  excess  that  the  protective  bodies  are  formed  in  amounts  not 
only  sufficient  to  meet  the  requirements,  but  in  such  excess  as  to  free 
circulating  immune  substances  in  the  blood.  This  hypothesis  in- 
troduced an  entirely  new  terminology  into  the  subject.  It  was 
supposed  that  cells  normally  possess  certain  specific  receptors  or 
combining  groups  for  the  injurious  substances  much  as  a  struc- 
tural chemical  formula  exhibits  free  valencies  on  the  part  of  certain 
elements  or  groups.  When  all  these  combining  groups  of  the  cell 
are  utilized  and  uncombined  poisonous  material  exists  in  the  circula- 
tion, the  cell  produces  and  liberates  additional  receptors  even  in 
excess  of  demand.  These  free  receptors  constitute  the  circulating 
immune  substances.  The  study  of  immune  substance  demon- 


GENERAL  PHENOMENA  OF  IMMUNITY  27 

strates  somewhat  variable  activities.  For  example,  it  was  found 
that  antitoxins  operate  differently  from  other  immune  substances; 
that  agglutinins  and  precipitins  operate  in  a  special  fashion  which 
is  practically  identical  for  both  substances ;  and  that  cytolysins,  includ- 
ing the  lytic  bodies  for  bacteria  as  well  as  for  animal  cells,  require 
the  presence  of  fresh  serum  containing  the  so-called  complement  or 
alexin.  The  specific  cytolysins  were  found  to  be  similar  to  certain 
other  substances  which  are  now  referred  to  as  complement-fixing 
bodies.  Finally  the  discovery  of  opsonins  and  tropins  showed  that 
there  is  in  all  probability  a  fourth  group  or  sub-group  of  these 
immune  substances. 

The  Ehrlich  Classification. — Ehrlich,  on  the  basis  of  the  general 
outline  given  above,  divided  the  immune  bodies  into  three  groups, 
depending  upon  demonstrable  differences  in  their  nature.  He  found 
that  the  receptors  in  some  instances  are  not  immunologically  simple 
bodies,  but  that  even  in  this  sense  they  show  varying  degrees  of 
complexity.  In  the  more  complex  forms  the  actual  receptor  or 
combining  groups  constitute  only  a  part  of  the  immune  substances, 
and  he  therefore  applied  a  more  comprehensive  term,  the  haptines. 
•He  included  in  the  haptines  of  the  first  order  the  antitoxins,  in  the 
haptines  of  the  second  order  the  agglutinins  and  precipitins,  and  in 
the  haptines  of  the  third  order  the  cytolysins  and  other  amboceptors. 
The  early  studies  of  antitoxins  made  it  appear  that  the  neutralizing 
effect  of  these  substances  was  similar  to  the  neutralizing  action  of 
alkalies  and  acids,  but  it  was  subsequently  discovered  that  such 
combinations  may  be,  at  least  in  part,  dissociated.  It  was  then 
found  that  toxin  may  undergo  a  variety  of  changes  as  the  result  of 
preservation.  Subsequently  it  was  learned  that  the  antitoxin  would 
combine  not  only  with  the  toxin,  but  with  its  degeneration  products. 
This  has  complicated  the  conception  considerably,  and  we  may  say 
in  brief  that  according  to  the  Ehrlich  conception  the  antitoxin  con- 
stitutes a  simple  receptor  or  combining  group  capable  of  entering 
into  combination  with  a  special  combining  group  in  the  toxin  called 
the  haptophore.  In  order  to  account  for  the  combination  of  anti- 
toxin with  the  degeneration  products  of  toxins  it  was  necessary  to 
assume  that  the  toxin  exhibits  its  essential  combining  property  in 
the  haptophore  group  and  that  the  toxin  also  possesses  a  toxophore 
group,  serving  to  give  it  its  poisonous  character  and  partly  de- 
stroyed during  preservation  or  by  certain  degrees  of  heat.  The 
second  category  of  Ehrlich  includes  the  agglutinins  and  precipitins. 
In  the  study  of  these  substances  it  was  found  that  the  agglutinins 
and  precipitins  may  be  deprived  of  the  agglutinating  and  precipitat- 
ing properties  by  preservation  or  by  the  application  of  certain  de- 
grees of  heat.  Ehrlich,  therefore,  conceived  the  idea  that  in  this 
instance  we  have  to  deal  with  a  somewhat  more  complicated  haptine 
containing  a  combining  group  and  a  so-called  zymophore  group, 
the  latter  leading  to  the  special  reaction.  These  constitute  the  re- 
ceptors or  haptines  of  the  second  order.  This  assumption  is  neces- 


28  THE  PRINCIPLES  OF  IMMUNOLOGY 

sary,  because  even  although  the  agglutinating  and  precipitating 
properties  are  destroyed  by  heat  or  other  means,  nevertheless,  there 
remains  a  group  capable  of  entering  into  combination  with  the  anti- 
genie  substances,  so  that  the  addition  of  a  complete  agglutinin  or 
precipitin  produces  no  effect.  The  third  category  of  Ehrlich  includes 
the  receptor  or  haptine  which  has  been  named  by  Ehrlich  the  ambo- 
ceptor,  and  by  Bordet  the  sensitizer.  In  this  instance  the  receptor 
produced  by  the  cell  is  conceived  as  a  body  possessing  two  com- 
bining groups,  one  serving  to  combine  with  the  antigen  and  the 
other  serving  to  combine  with  complement.  These  two  groups  have 
been  called  the  cytophilic  group  and  the  complementophilic  group. 
The  complement  is  a  thermolabile  substance  which  has  little  or  no 
capacity  for  combining  with  the  antigen.  Accepting  Ehrlich's  hy- 
pothesis, this  haptine  of  the  third  order  constitutes  an  intermediary 
body  through  the  action  of  which  the  complement  is  brought  into 
contact  with  the  cells,  be  they  bacterial  or  animal,  so  as  to  lead  to 
solution.  Bordet,  however,  believes  that  the  immune  body  enters 
directly  into  combination  with  the  antigen,  thereby  "  sensitizing  " 
it  so  that  the  complex  is  operated  upon  by  the  complement,  or  as  he 
calls  it,  the  alexin.  The  discovery  of  the  phenomenon  of  comple- 
ment-fixation demonstrated  that  a  similar  substance  may  operate  in 
the  presence  of  dissolved  protein  and  complement,  so  as  to  engage 
the  complement  in  such  a  fashion  that  it  is  not  available  for  other 
reactions.  In  these  reactions  the  participation  of  the  complement  is 
an  essential  and  necessary  condition  of  the  reaction.  The  original 
Ehrlich  theory  could  not  consider  the  subsequently  discovered 
opsonin  or  tropin.  This  substance  prepares  bacteria  and  other 
cells  for  phagocytosis.  It  was  at  first  supposed  to  be  a  simple 
immune  substance,  but  as  the  study  of  its  activity  progressed  it  was 
found  that  the  presence  of  complement  increases  its  activity,  al- 
though this  latter  body  is  not  essential  and  necessary.  We,  there- 
fore, propose  to  consider  the  opsonin  as  belonging  essentially  to  the 
haptines  of  the  third  order.  The  way  in  which  this  differs  from  the 
original  haptine  of  the  third  order  is  simply  in  the  fact  that  com- 
plement may  or  may  not  be  utilized  in  the  reaction.  In  order  to 
differentiate  we  suggest  that  the  amboceptors  of  Ehrlich  be  looked 
upon  as  "  obligate  "  amboceptors  and  the  opsonin  be  regarded  as  a 
"  facultative  "  amboceptor. 

Recent  Criticism  of  the  Ehrlich  Hypothesis. — Following  the 
earlier  discoveries  of  immune  phenomena  numerous  studies  were 
made  of  the  chemistry  of  immune  substances  and  immune  reactions. 
These  will  be  discussed  in  the  chapters  on  the  special  immune  reac- 
tions. It  may  be  said  at  this  time  that  many  objections  have  been 
raised  to  the  Ehrlich  hypothesis,  particularly  as  the  study  of  physi- 
cal chemistry,  more  particularly  that  part  which  refers  to  colloids, 
has  advanced.  As  will  be  seen  from  the  brief  review  of  the  Ehrlich 
hypothesis  given  above,  this  investigator  was  much  influenced  by 
the  status  of  chemistry  which  prevailed  when  he  announced  his 


GENERAL  PHENOMENA  OF  IMMUNITY  29 

views.  The  idea  predominated  that  the  immune  reactions  resemble 
the  more  or  less  fixed  changes  which  are  seen  in  the  chemical  reac- 
tions of  crystalloids.  As  it  was  found  that  practically  all  immune 
substances  are  colloidal  in  nature  and  either  are  proteins  or  are  very 
closely  related  to  proteins  the  similarity  of  the  immune  reactions  to 
colloidal  reactions  became  more  and  more  strongly  emphasized.  In 
fact,  in  a  general  way,  practically  all  immune  reactions  parallel  in 
their  general  phases  similar  demonstrable  reactions  with  colloids. 
There  is  but  one  feature  of  immune  reactions  which  has  not  yet 
been  explained  on  the  basis  of  colloid  chemistry,  namely,  their 
specificity.  This  does  not  mean,  however,  that  further  investiga- 
tion will  not  clear  up  this  phase  of  the  problem.  It  is  but  fair  to  say 
that  the  Ehrlich  hypothesis  provides  an  excellent  basis  for  the  classi- 
fication of  immune  phenomena,  but  as  will  be  shown  subsequently, 
the  conception  underlying  the  Ehrlich  hypothesis  is  not  adapted  to 
the  more  modern  views  of  the  mechanism  of  immune  reactions.  The 
combination  of  toxin  and  antitoxin  shows  numerous  features  not  to 
be  explained  by  the  simpler  reactions  of  crystalloids.  The  same  is 
true  of  agglutination,  precipitation,  cytolysis,  complement-fixation, 
and  anaphylaxis. 

Specificity  of  Immune  Reactions. — The  antitoxin  elaborated  in 
the  response  to  injections  of  diphtheria  toxin  or  to  the  presence  of 
the  disease  itself  is  a  substance  which  reacts  only  with  diphtheria 
toxin.  The  agglutinins  and  precipitins  produced  by  injection  of 
bacteria  and  of  dissolved  proteins  act  most  powerfully  upon  the  sub- 
stances used  for  injection.  In  this  case,  however,  these  immune 
bodies  may  also  react  less  strongly  with  other  closely  related  bac^ 
teria  or  proteins.  Cytolysins  induced  by  the  injection  of  certain 
cells  react  strongly  with  those  cells,  but  also  less  strongly  with 
closely  related  cells.  This  phenomenon  of  reaction  with  closely  re- 
lated bodies  is  spoken  of  as  the  group  phenomenon  and  may  be 
exhibited  also  in  connection  with  complement  fixation  and  ana- 
phylaxis. Even  where  purified  proteins  are  employed  the  same 
phenomenon  may  be  observed.  In  spite  of  the  group  reaction,  how- 
ever, the  immune  substances  are  most  highly  specific  for  their  spe- 
cial antigens.  Specificity  has  been  employed  for  the  detection  of 
particular  proteins  of  animal  species,  of  bacterial  species,  and  it 
has  lent  support  to  the  Darwinian  theory  of  species  relationship  and 
evolution.  Much  thought  and  study  has  been  given  to  the  resem- 
blance between  immune  substances  and  enzymes,  but  in  no  sense 
can  enzymes  be  said  to  have  the  same  specific  character  as  immune 
bodies.  There  is  no  satisfactory  explanation  of  specificity.  Why 
the  injection  of  red  blood-corpuscles  of  the  sheep  should  induce  the 
formation  of  a  hemolysin  capable  of  dissolving  the  red  cells  of  the 
sheep  but  not  of  other  animals,  except  in  minor  degree  of  those 
closely  related  to  the  sheep,  cannot  be  explained.  As  can  readily  be 
understood,  specificity  involves  the  use  of  special  antigens  and  the 
formation  of  more  or  less  specific  immune  substances.  The  wide 


30  THE  PRINCIPLES  OF  IMMUNOLOGY 

range  of  possibility  in  this  connection  is  indicated  in  the  building- 
stone  theory  of  Abderhalden.  Bearing  in  mind  that  practically  all 
immune  substances  are  protein  in  nature  and  that  proteins  are  made 
up  of  numerous  amino-acids,  Abderhalden  calculated  that  twenty 
amino-acids  could  be  so  combined  as  to  form  2,432,902,008,176,640,000 
different  compounds.  He  illustrates  this  possibility  by  stating  that 
if  three  amino-acids  are  building  stones  which  may  be  designated 
A,  B,  and  C,  they  can  be  grouped  together  so  as  to  form  six  different 
combinations,  ABC,  ACB,  BCA,  BAC,  CAB,  and  CBA,  and  that 
four  building  stones  can  form  twenty-six  such  combinations  and  so 
on  until  the  enormous  possibility  of  different  combinations  of 
twenty  amino-acids  is  reached,  as  illustrated  in  the  figures  given 
above.  Chemically  no  such  enormous  number  of  proteins  is  known, 
but  if  immune  specificity  could  be  shown  to  depend  upon  slight 
differences  of  molecular  arrangement,  Abderhalden's  figures  indi- 
cate the  number  of  immunologically  specific  proteins  obtainable. 
Taking  for  granted  the  phenomenon  of  specificity,  that  of  the  group 
reactions  can  be  more  readily  explained.  In  this  case  it  is  assumed 
that  in  the  proteins  of  closely  related  species  there  is  some  group  of 
molecules  common  to  these  species,  and  further  that  the  formation 
of  immune  substances  in  response  to  injections  of  this  common 
group  leads  to  the  production  of  a  substance  which  may  react  with 
the  common  group.  In  each  species,  however,  there  is  in  addition 
to  the  common  group  special  groups  which  determine  the  specificity 
of  the  substance  as  an  antigen  as  well  as  the  production  of  an  im- 
mune substance  with  a  higher  degree  of  affinity  for  the  combined 
groups  of  the  particular  species  than  for  the  common  group. 

Non-specific  Therapy  of  Infectious  Disease. — As  a  result  of  the 
extensive  studies  of  infectious  disease  various  modes  of  treatment 
have  been  elaborated.  It  is  well  understood  that  the  organism 
offers  resistance  to  these  infections  and  that  the  support  of  circula- 
tion and  excretion  by  simpler  pharmacological  methods  aids  mate- 
rially in  the  treatment.  Not  only  is  this  true,  but  the  investigation 
of  various  drugs  has  determined  the  specific  chemo-therapeutic  treat- 
ment of  infections.  Examples  of  this  are  seen  in  the  use  of  quinine 
in  malaria,  arsenic  in  trypanosomiasis  and  spirochetosis,  and  of 
emetin  in  amebiasis.  The  treatment  based  more  particularly  upon 
immunological  methods  has  been  largely  specific,  but  more  recent 
studies  have  given  encouragement  in  the  use  of  certain  non-specific 
methods  of  treatment.  It  was  found,  for  example,  that  the  use  of 
typhoid  vaccine  is  of  value  not  only  in  the  treatment  of  typhoid 
fever,  but  in  other  diseases,  and  typhoid  vaccines  either  in  the  form 
of  the  usual  killed  organisms  or  organisms  sensitized  with  specific 
immune  sera  have  produced  beneficial  results  in  such  diseases  as 
acute  articular  rheumatism,  sub-acute  and  chronic  arthritis,  and  in 
certain  other  infections.  Similarly  the  use  of  blood  serum,  of  pure 
proteins,  of  leucocyte  extracts,  of  fibrin  derivatives,  and  of  certain 
other  protein  derivatives  has  appeared  to  be  beneficial.  It  is  not  to 


GENERAL  PHENOMENA  OF  IMMUNITY  31 

be  assumed  that  this  method  of  non-specific  treatment  is  of  con- 
clusively proven  value,  but  the  effects  observed  in  a  certain  per- 
centage of  cases  offers  the  hope  that  the  method  may  be  so  perfected 
as  to  give  improved  results. 

The  general  reaction  following  subcutaneous  injection  of  these 
substances  may  or  may  not  be  severe,  but  if  they  are  administered 
by  the  intravenous  route  the  reaction  is  likely  to  be  pronounced. 
Frequently  a  chill  appears  and  almost  all  cases  develop  fever  which 
may  be  very  high.  Sometimes  there  is  a  general  feeling  of  discom- 
fort associated  with  headache  and  nausea.  In  typhoid  fever  it  is 
reported  that  hemorrhages  not  infrequently  occur  as  the  result  of 
the  therapeutic  use  of  sensitized  and  of  non-sensitized  typhoid  vac- 
cine. This  does  not  appear  in  other  diseases,  and  although  pro- 
tein substances  and  their  cleavage  products,  upon  injection,  tend  to 
decrease  the  coagulation  time,  yet  the  use  of  blood  serum  in  the  treat- 
ment of  hemophilia  often  has  a  favorable  effect  in  preventing  hemor- 
rhage. In  addition  to  the  possibility  of  hemorrhage  in  typhoid  fever 
there  are  definite  contraindications  to  this  form  of  therapy  in  preg- 
nancy, in  patients  with  organic  heart  disease,  and  in  those  with  high 
blood-pressure.  The  influence  of  this  non-specific  method  of  treat- 
ment is  not  clearly  understood.  The  question  as  to  whether  or  not 
the  known  forms  of  antibodies  are  liberated  or  stimulated  has  been 
studied  by  numerous  workers  with  contradictory  results.  Some 
have  found  an  increase  of  agglutinins  and  precipitins  for  the  specific 
organisms  concerned  in  the  disease,  following  non-specific  protein 
injections,  but  this  is  contradicted  by  other  workers.  Regardless  of 
the  question  of  stimulation  of  special  immune  bodies  it  is  important 
to  know  what  other  protective  influences  may  be  set  at  work. 

Fever  is  a  common  incident  of  the  injection  of  proteins  or  pro- 
tein products,  especially  when  they  are  given  intravenously.  This 
is  sometimes  accompanied  by  leucocytosis,  but  neither  leucocytosis, 
marked  acceleration  of  pulse-rate,  nor  the  other  clinical  accompani- 
ments of  fever  necessarily  appear.  It  has  been  demonstrated  that 
increased  temperature  aids  in  the  production  of  agglutinins  and  bac- 
teriolytic  substances.  In  most  instances  the  degree  of  temperature 
reached  in  fever  has  no  deleterious  effect  directly  upon  the  bacteria 
concerned,  except  possibly  in  the  case  of  infections  with  the 
gonococcus  and  with  the  spirochete  of  relapsing  fever.  It  has 
been  suggested  that  high  body  temperature  may  favor  the  com- 
bination of  the  antigen  and  immune  substances,  but  this  has  not 
been  conclusively  demonstrated.  The  injection  of  proteins  may  lead 
to  an  increase  in  the  number  of  circulating  leucocytes,  although  this 
is  not  invariably  the  case.  The  influence  of  such  a  hyper-leucocytosis 
in  combating  infection  is  at  least  partly  because  of  the  fact  that 
these  cells  ingest  and  destroy  bacteria.  Nevertheless,  certain 
infectious  diseases,  such  as  typhoid  fever,  may  run  their  course 
without  exhibiting  leucocytosis,  and  it  is  therefore  not  essential  for 
recovery  that  the  leucocytes  be  increased  in  number.  It  must  be 


32  THE  PRINCIPLES  OF  IMMUNOLOGY 

pointed  out,  however,  that  phagocytosis  is  not  the  only  way  in 
which  an  increase  of  leucocytes  may  operate  beneficially.  The 
studies  of  Hiss  and  Zinsser  indicate  that  extracts  of  leucocytes  have 
a  beneficial  effect  on  infections  and  others  have  confirmed  these 
results.  Bail  claims  that  a  fresh  emulsion  of  leucocytes  will  aid  in 
neutralization  by  the  specific  anti-serum  of  endotoxin  obtained  from 
cholera  vibrios.  Jobling  and  Bull,  however,  demonstrated  that  leu- 
coprotease  "  will  destroy  the  toxic  extracts  of  typhoid  bacilli  and 
meningococci,  and  it  is  not  improbable  that  a  similar  explanation 
will  apply  to  the  results  obtained  by  Bail." 

There  are  other  possible  changes  in  the  blood  as  the  result  of  the 
injection  of  protein.  The  work  of  Jobling  and  his  collaborators  has 
thrown  great  light  on  the  alterations  of  ferments  and  anti-ferments 
in  the  blood  under  a  wide  variety  of  conditions.  The  injection  of 
various  substances  is  almost  invariably  followed  by  a  considerable 
mobilization  of  the  serum  ferments,  more  particularly  the  protease, 
and  usually  also  the  esterase.  The  value  of  the  protease  is  probably 
in  the  direction  of  breaking  down  toxic  split-protein  products,  which 
probably  originate  during  the  course  of  infectious  disease,  as  the 
result  of  the  splitting  of  bacteria  and  perhaps  also  of  the  body  pro- 
teins. Protease  does  not  act  directly  upon  living  bacteria,  but  it  is 
to  be  considered  possible  that  the  esterase  may  break  up  the  lipoid 
or  lipoid-protein  surface  of  the  bacteria  and  therefore  aid  in  their 
destruction.  If  we  concede  that  the  toxic  protein  split  products  aid 
in  the  virulence  of  bacteria  it  is  possible  that  even  although  the 
protease  simply  breaks  down  these  products  into  simpler  non-toxic 
substances  and  does  not  directly  attack  the  bacteria,  yet  the  relief  to 
the  body  afforded  by  this  detoxifying  action  may  assist  it  more 
permanently  in  combating  disease.  In  certain  states,  such  as  preg- 
nancy, in  disease  such  as  cancer,  and  in  the  course  of  vaccine  treat- 
ment the  anti-ferment  titer  of  the  blood  has  been  found  to  be  high. 
Jobling  and  Peterson  found  that  the  anti-ferment  power  of  the  blood 
depends  upon  the  amount  of  unsaturated  lipoids  present  in  highly 
dispersed  phase  in  the  serum  and  Bogolemez  suggests  that  lipoids 
may  serve  to  inhibit  toxins,  as  is  true  in  relation  to  the  toxin  of 
bacillus  botulinus.  Anti-ferment  is  not  increased  following  protein 
injections  and  plays  no  part  in  the  non-specific  therapy  of  infectious 
disease,  but  inasmuch  as  the  change  may  be  seen  in  immune  states, 
such  as  that  following  vaccination,  it  may  be  of  importance  in  non- 
specific resistance  to  infection.  In  addition  to  the  changes  in  fer- 
ments Jobling  has  found  that  the  injection  of  non-specific  proteins 
may  produce  changes  in  the  viscosity  of  the  serum.  It  is  known 
that  if  precipitates  are  formed  in  serum  by  the  action  of  a  specific 
precipitating  serum,  conditions  favorable  to  protease  activity  are 
produced  and  the  changes  in  viscosity  produced  by  protein  injec- 
tions may  similarly  aid  proteolytic  activity.  These  changes  in 
ferment  content  and  physical  character  of  the  serum  are  of  short  dura- 
tion and  are  probably  contemporaneous  with  the  chill  and  fever. 


GENERAL  PHENOMENA  OF  IMMUNITY  33 

They  do  not  directly  account  for  permanent  improvement  seen  in 
many  patients,  but  if  they  rid  the  body  of  toxic  substances  for  a 
short  period  of  time  the  natural  resistance  may  thereby  become 
more  effective  than  would  otherwise  be  the  case. 

The  Site  of  Antibody  Formation. — Aside  from  a  few  fairly  well- 
established  facts  the  problem  as  to  exactly  where  antibodies  are 
formed  still  remains  obscure.  In  general  it  is  assumed  that  anti- 
bodies are  not  products  of  simple  inversions 'of  the  foreign  protein 
substances  parentally  introduced  or  as  particular  functions  of  spe- 
cial organs,  but  are  the  result  of  general  cell  reactions  on  the  part 
of  the  host.  Much  evidence  points  to  the  lymphatic  organs,  the 
spleen,  the  liver,  and  the  bone  marrow  as  places  where  antibody 
formation  is  most  active.  Metchnikoff  thought  that  antitoxins  and 
bacteriolysins  originate  in  the  lymphatic  organs  and  more  particu- 
larly in  the  spleen  and  the  bone  marrow.  Bordet  attempted  to 
show  that  bacteriolysins  are  derived  from  the  leucocytes.  Pfeiffer 
and  Mark  injected  dead  cholera  spirilla  into  animals,  exsanguinated 
these  five  days  after  the  injection,  and  found  the  antibodies  more 
concentrated  in  the  spleen  than  in  the  blood  serum  itself.  These 
authors  also  found  that  after  a  single  injection  of  these  organisms, 
the  spleen,  the  bone  marrow,  and  the  lymph-nodes  contained  the 
specific  antibodies  before  they  could  be  detected  in  the  blood,  and 
further  that  as  time  passed  these  tissues  became  less  active  in  spite 
of  the  fact  that  the  bacteriolysins  increased  in  the  blood.  Deutsch 
corroborated  these  findings  with  bacillus  typhosus  and  Castellani 
with  bacillus  dysenterise.  These  authors  agree,  however,  that  the 
spleen  is  not  essential,  since  its  removal  but  slightly  inhibits  the 
formation  of  antibodies,.  Hektoen's  experiments  demonstrated  that 
in  dogs  splenectomy  just  before  and  after  the  injection  of  alien 
blood-corpuscles  was  followed  by  a  much  lower,  but  otherwise  typi- 
cal antibody  curve,  than  is  usually  the  case  in  dogs  under  normal 
conditions.  London  also  reported  a  decreased  formation  of 
hemolysins  after  splenectomy,  but  this  work  has  been  contradicted 
by  Yakuschewitch.  Karsner,  Amiral,  and  Bock  found  that  splenec- 
tomy produces  no  change  in  hemopsonins  of  the  circulating  blood 
that  is  clearly  demonstrable  by  in  vitro  test,  and  that  the  blood  from 
the  spleen  is  no  richer  in  hemopsonins  than  is  blood  from  other 
organs.  Carrel  and  Ingebrigsten  have  produced  hemolysins  in  the 
growing  embryonic  spleen.  More  recently  Przygode  succeeded  in 
producing  precipitins  in  vitro  by  culture  of  splenic  tissue,  and  Miiller 
by  transplanting  splenic  tissue  from  guinea-pigs,  previously  injected 
with  sheep  corpuscles,  into  the  peritoneal  cavity  of  normal  guinea- 
pigs.  It  seems  to  us  that  since  the  spleen  is  an  organ  physiologically 
designated  for  the  destruction  of  erythrocytes  and  also  of  other 
foreign  substances  through  the  activity  of  its  hemophages,  splenic 
tissue  on  transplantation  will  carry  with  it  much  antigenic  sub- 
stance. Whether  or  not  these  hemophages  participate  in  antibody 
production  is  at  present  difficult  to  say. 
3 


34  THE  PRINCIPLES  OF  IMMUNOLOGY 

For  rapid  production  of  antibodies  Violle  injected  organisms 
directly  into  the  gall-bladder.  This  fact  is  of  interest  because  it 
indicates  a  possible  function  of  the  liver  in  the  production  of  immune 
bodies.  Miiller  claims  to  have  been  able  to  stimulate  the  formation 
of  hemolysin  in  liver  tissue  suspended  in  Ringer's  solution  outside 
the  animal  body.  By  perfusing  the  organ  with  solutions  contain- 
ing iodine  (iodipin)  the  effect  was  augmented,  and  he  believes  that 
in  the  normal  animal  the  iodine  of  the  thyroid  may  play  a  certain 
role  in  stimulating  this  special  activity  of  the  liver.  Gay  and  Rusk 
found  no  evidence  to  uphold  the  supposed  influence  of  iodipin. 
Hektoen  and  Carlson  believe  that  both  the  spleen  and  liver  are  equally 
concerned  in  antibody  formation,  but  Hektoen  and  Curtis  found 
that  in  rats  removal  of  about  one-half  of  the  liver  appears  to  have 
no  effect  on  the  development  of  hemolysin  for  sheep  corpuscles. 
The  liver,  just  as  the  spleen,  possesses  highly  active  phagocytic 
endothelial  cells  which  may  play  an  important  role  in  the  produc- 
tion of  antibodies. 

Numerous  authors  have  shown  that  agglutinins  appear  in  the 
blood  stream  before  they  are  present  in  the  extracts  of  any  organ. 
The  question,  however,  of  whether  or  not  the  leucocytes  are  in- 
volved in  this  generative  process  is  a  matter  of  considerable  contro- 
versy. Achard  and  Bensaud  and  others  controvert  the  leucocytic  or 
local  origin  of  agglutinins,  whereas  Cantacuz^ne  and  also  Swerew 
support  this  local  origin  in  the  formation  of  precipitins ;  they  noted 
a  hypoleucocytosis  followed  by  a  marked  hyperleucocytosis,  which 
they  think  is  responsible  for  the  liberation  of  precipitins.  Petit  and 
Carlson,  Vaughan,  Cumming,  and  McGlumphy  found  that  sub- 
stances like  egg-white  and  serum  disappear  quickly  from  the  cir- 
culating blood ;  in  fact,  within  a  few  hours  after  the  introduction  of 
these  substances.  Gay,  however,  has  shown  by  means  of  comple- 
ment-fixation that  even  in  immune  animals  such  antigens  are  dem- 
onstrable after  twenty-four  hours,  but  not  after  forty-eight  hours. 
It  was  not  possible  to  demonstrate  the  antigen  by  the  fixation  method 
in  organs  like  spleen,  lymph-nodes,  liver,  kidney,  and  muscles, 
either  at  the  time  antigen  was  present  in  the  blood  or  twenty-four 
hours  thereafter.  That  the  cells  lining  the  blood-vessels  may  have 
certain  powers  of  antibody  production  may  be  shown  by  the  fact 
that  a  blood-vessel  from  an  animal  which  has  received  several  injec- 
tions of  sheep  erythrocytes  and  which  has  been  dissected  out  soon 
after  death  of  the  animal  and  washed  free  from  blood,  has  the  power 
to  hemolyze  a  suspension  of  fresh,  non-sensitized  sheep  cells  (Van 
Calcar).  Kraus  and  Levaditi  furthermore  have  shown  that  there 
exists  a  certain  relationship  between  precipitins  and  the  number  of 
circulating  leucocytes.  Acute  loss  of  blood  profoundly  affects  anti- 
body production.  The  earliest  observations  seem  to  have  been  those 
of  Roux  and  Vaillard.  They  found  that  in  horses  actively  im- 
munized against  tetanus  toxin,  bleeding  causes  a  drop  in  the  anti- 
toxin content  in  the  blood,  followed  by  a  sharp  rise  in  a  short  time. 


GENERAL  PHENOMENA  OF  IMMUNITY  35 

By  continuous  daily  bleedings  Hahn  and  Langer  recently  succeeded 
in  increasing  the  agglutinin  content  250,000  times  its  original  value. 
Similarly  Madsen  and  Tallquist  have  shown  that  certain  poisons 
which  destroy  erythrocytes  may  increase  the  production  of  anti- 
bodies possibly  by  the  action  of  the  same  mechanism  as  that  whereby 
hemorrhage  stimulates  antibody  formation.  Rusk  has  found  that 
animals  intoxicated  with  benzol  produce  hemolysins  and  precipitins 
much  less  efficiently  than  normal  animals.  Since  benzol  affects 
particularly  the  bone  marrow  and  the  lymphatic  apparatus,  this 
evidence  points  in  favor  of  the  view  that  these  tissues  are  largely 
involved  in  the  production  of  hemolysins  and  of  precipitins. 

According  to  Hektoen  and  Curtis,  adrenalectomy  in  normal  dogs 
and  in  dogs  at  the  height  of  the  antibody  curve  after  the  injection 
of  rat  corpuscles  does  not  cause  a  decrease  in  the  antibody  content 
of  the  blood  serum.  Gates  was  able  to  remove  approximately  three- 
quarters  to  seven-eighths  of  the  adrenal  tissue  of  guinea-pigs  with- 
out causing  symptoms  of  adrenal  insufficiency.  Guinea-pigs  thus 
treated  were  injected  with  bacillus  typhosus  and  with  hen  cor- 
puscles, and  the  results  demonstrated  that  adrenalectomy  had  no 
influence  upon  the  rise  or  persistence  of  antibodies  in  the  blood, 
and  therefore  the  adrenals  appear  to  play  no  essential  part  in  the 
mechanism  of  antibody  production. 

The  results  of  Tjeldstad  had  shown  that  thyroidectomy  failed  to 
influence  antibody  production.  Similar  observations  were  recorded 
by  Hektoen  and  Curtis,  and  others,  but  Frouin  was  more  conserva- 
tive in  his  conclusions,  and  Garibaldi  has  recently  renewed  an  in- 
terest in  this  matter  by  reporting  that  the  hemolytic  titer  of  the 
serum  of  thyroidectomized  rabbits  is  much  higher  than  that  of  his 
control  animals,  therefore  concluding  that  thyroidectomy  definitely 
favors  antibody  production. 

We  know  from  the  experiments  of  Was,sermann  and  Takaki  that 
brain  substance  neutralizes  tetanus  toxin,  but  this  fact  does  not 
indicate  this  organ  to  be  of  much  importance  in  the  production  of 
antibodies.  In  fact,  we  have  learned  from  the  experiments  of  Loewi 
and  Meyer  that  injection  of  toxin  into  the  nervous  system  produces  an 
increased  susceptibility  of  the  animal  rather  than  increase  of  resistance. 

Production  of  Antibodies  at  Site  of  Injection. — Certain  experi- 
ments indicate  that  antibodies  may  also  be  produced  at  the  place  of 
introduction  of  the  antigen.  Romer  and  also  von  Dungern  have 
shown  that  immunization  by  way  of  the  conjunctiva  or  anterior 
chamber  of  the  eye  results  in  the  formation  of  antibodies  in  the 
aqueous  humor  before  they  can  be  demonstrated  in  the  blood.  These 
experiments  also  demonstrated  that  the  opposite  eye  produces  no 
antibody.  Wassermann  and  Citron  ligated  a  rabbit's  ear  at  its  base 
after  a  subcutaneous  injection  of  bacteria.  The  ligation  was  left 
for  several  hours,  and  after  nine  days  the  bactericidal  titer  of  the 
blood  serum  determined  and  the  ear  amputated.  An  immediate  and 
rapid  drop  of  antibody  in  the  blood  which  occurred  after  the  ampu- 


36  THE  PRINCIPLES  OF  IMMUNOLOGY 

tation  indicates  that  the  main  source  of  antibody  formation  was 
removed  or  the  absorption  of  the  ahtigenic  substances  entirely 
stopped.  Forsmann  and  Lundstrom  studied  the  curve  of  pro- 
duction of  botulinus  antitoxin  following  single  intravenous  or 
subcutaneous  injection  of  the  toxin.  The  curve  following  the 
subcutaneous  injection  reached  its  highest  level  on  the  fifteenth  day, 
while  that  following  the  intravenous  injection  attained  the  maximum 
on  the  tenth  day.  It  must  be  inferred  that  the  subcutaneous  method 
of  injection  introduces  the  factor  of  slow  absorption,  but  it  is  also 
possible  that  some  local  factor  may  enter  into  the  phenomenon. 
Immunization  of  horses  with  diphtheria  toxin  results  in  a  greater 
yield  of  antitoxin  when  the  horses  are  injected  subcutaneously,  but 
this  does  not  necessarily  prove  a  local  production  of  antitoxin  at  the 
site  of  inoculation.  Local  cellular  participation  in  immune  reactions 
will  be  discussed  further  in  the  chapter  on  hypersusceptibility. 
There  is  little  doubt  that  local  reactions  are  of  significance  and  that 
absorption  may  be  influenced  by  local  changes.  The  production  of 
circulating  antibodies  in  any  considerable  amounts  undoubtedly  re- 
quires more  extensive  cellular  activity  than  that  about  the  site  of 
local  inoculations. 


CHAPTER  IV 
TOXINS  AND  ANTITOXINS 


GENERAL  NATURE  OF  TOXINS. 
THE  BACTERIAL  TOXINS. 

CLASSIFICATION. 
PATHOLOGICAL  EFFECTS. 
FORMATION  OF  ANTITOXINS. 

TECHNIC  OF  PRODUCING  DIPHTHERIA  TOXIN. 
THE  NATURE  OF  ANTITOXINS. 

STANDARDIZATION    OF   DIPHTHERIA   ANTITOXIN. 
THE  MINIMUM  LETHAL  DOSE    (M.L.D.). 
THE  L0    DOSE   (LIMES  NULL). 
THE  L+    DOSE   (LIMES  DEATH). 
TITRATION  OF  DIPHTHERIA  ANTITOXIN. 
THE  TOXIN  ANTITOXIN  UNION. 
THEORIES  OF  UNION. 
EHRLICH   THEORY. 
LAW   OF    MASS   ACTION. 

THE  DANYSZ  EFFECT. 

THERAPEUTIC  USE  OF  DIPHTHERIA  ANTITOXIN. 
VALUE. 

MODES  OF  ADMINISTRATION. 
NATURAL  IMMUNITY  TO  DIPHTHERIA. 

THE   SCHICK   TEST. 
ACTIVE  IMMUNIZATION  IN  MAN. 
TETANUS  TOXIN  AND  ANTITOXIN. 
TETANOTOXIN. 
TETANOSPASMIN. 
ROUTE  OF  ABSORPTION  OF  TOXIN. 
THERAPEUTIC  USE  OF  TETANUS  ANTITOXIN. 
PROPHYLAXIS. 

TREATMENT   OF  THE  DISEASE, 
DYSENTERY  TOXIN. 

THERAPEUTIC    USE   OF   DYSENTERY   ANTISERA. 
BOTULINUS  TOXIN. 

THE  USE  OF  IMMUNE  SERA  IN  BOTULISM. 
GAS  BACILLUS  TOXINS. 

THE  USE  OF  IMMUNE  SERA  IN  GAS   GANGRENE. 
TREATMENT  OF  THE  DISEASE. 
PROPHYLAXIS. 
BACTERIAL   HEMOTOXINS. 

STAPHYLOLYSIN  AND  ANTILYSIN. 
THE  PHYTOTOXINS. 
RICIN. 
ABRIN. 
CROTIN. 
CURCIN. 
PHASIN. 
THE    ZOOTOXINS. 

SNAKE  VENOMS. 

SCORPION  AND  SPIDER  POISON. 

CENTIPEDE  POISON. 

BEES,  WASPS,  AND  HORNETS. 

TOADS,  FROGS,  AND  SALAMANDERS. 

POISONOUS  FISH. 

EEL  SERUM. 

PARASITIC  PROTOZOA. 

MAMMALIA. 

ANIMAL   SERA. 

37 


38  THE  PRINCIPLES  OF  IMMUNOLOGY 

General  Nature  of  Toxins. — Toxins  are  soluble  poisonous  prod- 
ucts of  life  processes,  which  on  injection  into  animals  lead  to  the 
formation  of  antitoxins.  A  corollary  of  this  definition  sometimes  in- 
sisted upon  is  that  the  injurious  effect  of  these  toxic  bodies  must  be 
preceded  by  an  incubation  period,  but  in  certain  instances  this  in- 
cubation time  is  a  matter  of  minutes  or  hours,  as  is  the  case  with 
certain  snake  poisons.  The  toxins  are  divided  according  to  their 
origin  into  phytotoxins,  produced  by  vegetable  life,  and  zootoxins, 
produced  by  animal  life.  The  most  important  of  the  phytotoxins 
are  the  bacterial  toxins,  but  the  group  includes  also  ricin,  abrin, 
crotin,  robin,  and  curcin.  Certain  of  the  higher  plant  poisons  which  pro- 
duce the  varieties  of  "  hay  fever  "  in  susceptible  individuals  were 
formerly  considered  as  toxins,  but  this  view  has  now  been  discarded. 
The  poison  of  rhus  toxicodendron  (poison  ivy)  and  of  rhus  diversiloba 
(poison  oak)  might  be  considered  a  phytotoxin,  but  is  chemically  a  glu- 
coside  and  does  not  produce  antitoxin.  The  poisoning  of  non-edible 
mushrooms  is  due,  in  the  case  of  amanita  muscaris  and  helvella  escul- 
enta,  to  definite  chemical  compounds,  muscarine  and  helvellic  acid, 
which  do  not  produce  antibodies.  In  the  case  of  amanita  phalloides 
there  are  two  substances  of  toxic  nature,  a  thermolabile  hemolytic 
glucoside  capable  of  producing  an  anti-hemolysin,  and  a  thermo- 
stabile  toxin  of  unknown  composition  and  incapable  of  producing  a 
definite  immune  body.  The  most  important  of  the  zootoxins  are  the 
snake  venoms,  but  this  group  also  includes  the  poisons  of  spiders, 
scorpions,  centipedes,  bees,  wasps,  hornets,  dermal  glands  of  toads 
and  salamanders,  various  sera,  notably  that  of  the  eel,  and  certain 
poisonous  fish. 

Nicolle,  Cesari,  and  Jouan  divide  the  toxins  into  those  that  ap- 
pear in  the  form  of  definite  secretions,  as  snake  venoms,  those  which 
are  determined  by  logical  inference,  as  the  toxins  in  microbial  fil- 
trates, and  those  which  are  obtained  by  simple  maceration,  expres- 
sion, grinding,  or  autolysis,  the  endotoxins.  Experiments  with  the 
endotoxins  are  performed  in  large  part  with  the  microbial  bodies, 
and  therefore  these  workers  refer  to  the  endotoxins  as  solid  toxins. 

The  bacterial  toxins  are  synthetic  products  of  the  life  of  the  organ- 
isms themselves.  It  was  thought  for  years  that  the  bacteria  could 
synthesize  the  protein  toxin  from  simple  nitrogen-containing  com- 
pounds. More  modern  studies  oppose  this  view  and  state  that  more 
complex  substances,  such  as  proteoses  and  polypeptids,  are  essen- 
tial. It  seems  certain  that  nothing  less  complex  than  the  amino- 
acids  can  be  synthesized,  and  recent  studies  indicate  that  diphtheria 
toxin  is  not  a  synthetic  product,  but  rather  a  catabolic  substance 
elaborated  by  the  bacteria  only  in  the  presence  of  amino-acids  and 
certain  additional  substances,  probably  of  the  nature  of  vitamines. 
They  are  thus  to  be  distinguished  from  the  ptomains,  which  al- 
though products  of  bacterial  growth,  are  in  reality  formed  from  the 
culture  medium  and  vary  according  to  the  medium  rather  than 
according  to  the  organism.  The  chemical  nature  of  the  bacterial 


TOXINS  AND  ANTITOXINS  39 

toxins  is  uncertain,  but  they  appear  to  be  more  closely  related  to  the 
proteins  than  to  any  other  known  substance.  They  diffuse  through 
membranes  less  slowly  than  do  proteins,  and  therefore  are  pre- 
sumed to  have  a  smaller  molecular  size.  On  the  other  hand,  they 
are  digested  less  readily  than  proteins.  Like  proteins  they  are 
electro-positive  colloids,  and  are  precipitated  by  protein  precipitat- 
ing agents,  such  as  ammonium  sulphate.  As  against  this  is  the 
statement  that  toxins  may  be  so  far  purified  that  they  do  not  give 
the  protein  reactions.  They  resemble  enzymes  in  that  both  are 
colloids,  both  thermolabile,  dialyze  with  difficulty,  lose  strength  in 
passing  through  porcelain  filters,  resist  drying  and  dry  heat,  resist 
low  temperatures,  both  produce  antibodies,  both  deteriorate  after 
standing  in  solution  with  loss  of  zymophore  group,  but  without  loss 
of  haptophore  or  combining  groups.  The  difficulty  of  establishing 
the  toxins  as  enzymes  lies  in  the  fact  that  neither  toxin  nor  enzymes 
have  been  isolated  in  the  pure  state.  Furthermore,  they  do  not  act 
according  to  the  same  chemical  laws,  the  enzyme  operating  re- 
peatedly to  produce  a  large  effect  in  the  course  of  time  and  the  toxin 
acting  in  almost  direct  proportion  to  its  quantity.  In  summary  we 
may  quote  Oppenheimer  as  saying  of  toxins  that,  "  we  must  be 
contented  to  assume  that  they  are  large  molecular  complexes,  probably 
related  to  the  proteins,  corresponding  to  them  in  certain  properties, 
but  standing  even  nearer  to  the  equally  mysterious  enzymes  with 
whose  properties  they  show  the  most  extended  analogieG,  both  in 
their  reactions  and  in  their  activities." 

Toxins  may  be  injured  in  a  variety  of  ways.  They  may  be  de- 
stroyed, with  certain  exceptions,  by  moist  heat  at  about  80°  C.,  and 
resist  dry  heat  to  over  100°  C.  Light  operates  in  a  general  way 
according  to  its  intensity  and  penetrating  power  and  the  action  is 
intensified  by  the  presence  of  oxygen.  Diffuse  daylight  operates 
slowly,  but  direct  sunlight,  X-ray,  and  ultra-violet  rays  more  rapidly. 
They  are  destroyed  by  fluorescent  substances.  Oxygen  and  oxi- 
dizing substances  injure  and  destroy  toxins  both  in  vivo  and  in  vitro. 
Certain  chemicals  are  injurious,  as  the  salts  of  bivalent  and  trivalent 
metals,  but  not  of  monovalent  metals.  Certain  toxins,  particularly 
dysentery  and  diphtheria,  may  be  rendered  non-toxic  by  acids  and 
restored  to  toxicity  by  alkali.  They  may  be  bound  by  fats  and 
lipoids,  as  illustrated  in  part,  at  least,  by  the  neutralization  of 
tetanus  toxin  by  brain  substances.  Enzymes,  such  as  pepsin  and 
pancreatic  juice,  as  well  as  bile,  destroy  certain  toxins,  so  that  they 
produce  no  symptoms  following  ingestion,  the  striking  exception 
being  botulinus  toxin.  The  action  of  digestive  ferments  upon  toxins 
has  recently  been  studied  in  detail  by  Loewi.  He  finds  that  diph- 
theria toxin  is  destroyed  by  pepsin  and  ptyalin,  that  tetanus  toxin 
is  destroyed  by  trypsin  and  ptyalin,  but  not  by  pepsin ;  and  that 
dysentery  toxin  is  destroyed  by  the  action  of  the  duodenal  mucosa 
of  rabbits,  but  resists  digestion  with  trypsin,  ptyalin,  pepsin, 
and  papayotin. 


40  THE  PRINCIPLES  OF  IMMUNOLOGY 

Classification  of  Bacterial  Toxins. — The  bacterial  toxins  are 
classified  as  exotoxins  and  endotoxins,  the  former  appearing  in  the 
culture  medium  as  soluble  substances  and  the  latter  appearing  within 
the  bacterial  bodies.  These  intracellular  toxins  can  be  liberated  by 
digestion,  autolysis,  freezing  and  fine  grinding,  and  by  expression 
with  a  Buchner  press.  They  cause  the  symptoms  of  their  special 
diseases,  and  in  the  natural  course  of  the  disease  are  probably  liber- 
ated either  by  autolysis  or  by  the  action  of  the  enzymes  of  the  cells 
or  fluids  of  the  host.  The  tendency  to-day,  however,  is  to  accept 
the  view  that  the  so-called  endotoxins  are  not  produced  as  such,  but 
are  produced  from  the  bacteria  during  the  process  of  hydrolytic 
cleavage  of  the  bacterial  proteins  by  ferments  provided  by  the  host. 
Certain  bacteria,  such  as  diphtheria  and  tetanus,  produce  only  exo- 
toxins, whereas  the  typhoid  group  and  certain  other  organisms  were 
supposed  formerly  to  produce  only  endotoxins.  However,  Bull  has 
shown  that  certain  strains  of  the  gas  bacillus  of  Welch  produce  an 
active  exotoxin,  and  Ecker  has  shown  that  certain  strains  of  bacillus 
paratyphosus  B  produce  exotoxins,  and  this  has  been  confirmed  by 
Aronowitch.  Kraus  has  shown  a  similar  relationship  in  bacillus 
dysenteriae.  Studies  of  Admont  Clark  and  Felton  indicate  that  the 
strepto'coccus  hemolyticus  produces  a  filterable  toxic  product 
answering  all  the  requirements  of  a  true  toxin.  The  production  of 
exotoxins  is  important  for  practical  purposes  because  the  endo- 
toxins do  not  lead  to  antitoxin  formation  with  the  same  degree  of 
facility  as  do  exotoxins.  Recent  studies  by  Olitsky  and  Kligler  have 
shown  that  the  dysentery  bacillus  (Shiga)  produces  a  thermolabile 
exotoxin  and  a  thermostable  endotoxin,  the  latter  not  being  neutral- 
ized by  anti-exotoxic  serum.  A  potent  antiserum  for  both  toxins 
can  be  developed  in  the  horse.  The  exotoxin  appears  to  have  spe- 
cial affinity  for  the  nervous  system  of  the  rabbit  and  the  endotoxin 
operates  particularly  upon  the  intestine. 

Organisms  which  produce  exotoxins  show  a  considerable  variation  of 
this  property,  but,  on  the  whole,  such  toxins  are  more  virulent  and  more 
highly  antigenic  than  the  exotoxins  of  those  organisms  which  are  essen- 
tially endotoxin  producers.  Being  more  highly  antigenic  the  antitoxins 
produced  by  exotoxins  are  the  more  powerful,  as  is  well  known  in  the 
case  of  diphtheria  and  tetanus  antitoxins.  Nicolle,  Cesari,  and  Jouan 
maintain,  on  the  basis  of  certain  work  with  the  bacillus  of  Nocard,  that 
exotoxins  and  endotoxins  are  identical  in  the  case  of  a  given  organ- 
ism, but  the  more  recent  studies  of  Olitsky  and  Kligler  quoted  above 
would  indicate  that  this  is  not  true  of  dysentery  bacillus  (Shiga), 
and  therefore  not  a  general  law. 

The  exotoxins  include  diphtheria  toxin,  tetanus  toxin,  botulinus 
toxin,  dysentery  toxin,  paratyphoid  toxin,  and  bacillus  aerogenes 
capsulatus  (perfringens)  toxin.  Toxins  are  produced  also  by 
bacillus  edematiens  (Weinberg),  vibrion  septique,  bacillus  of  symp- 
tomatic anthrax,  bacillus  pyocyaneus,  streptococcus,  and  bacillus  in- 
fluenzse.  In  addition  it  is  claimed  by  Kolmer  and  his  co-workers 


TOXINS  AND  ANTITOXINS  41 

that  they  have  demonstrated  in  pneumonic  exudates  a  pneumococcus 
toxin.  A  number  of  other  organisms  produce  lytic  bodies  for  red 
blood-cells  or  hemotoxins,  such  as  staphylolysin  and  megath- 
eriolysin,  capable  of  inducing  the  formation  of  antilysins.  A  lytic 
body  for  leucocytes  is  also  produced  by  staphylococcus  aureus. 

The  pathological  effects  of  toxins  are  fundamentally  seen  in  the 
production  of  cloudy  swelling  and  even  fatty  degeneration  of  the 
parenchymatous  viscera,  heart,  vascular  muscle,  liver,  kidney,  and 
secreting  glands.  Local  inflammation  at  the  site  of  injection,  some- 
times leading  to  necrosis,  is  a  frequent  rinding.  Diphtheria  toxin 
may  show,  in  cases  of  paralysis,  myelin  sheath  degeneration  or  in- 
flammation of  nerves,  and  in  guinea-pigs  usually  shows  marked 
congestion  or  hemorrhage  in  the  adrenals.  Botulinus  toxin  leads  to 
meningeal  and  even  cerebral  thrombosis  and  small  hemorrhages. 
Both  botulinus  toxin  and  tetanus  toxin  have  a  marked  affinity  for 
the  nervous  system,  but  the  effects  are  seen  in  the  form  of  func- 
tional disturbance  rather  than  morphologically  demonstrable  change, 
except  for  the  vascular  changes  produced  by  botulinus  toxin. 

Formation  of  Antitoxins. — Antitoxins  are  produced  by  the  re- 
peated parenteral  injection  of  the  toxin.  Parenteral  injection  signi- 
fies introduction  into  the  body  by  routes  other  than  absorption 
through  the  alimentary  canal.  The  selection  of  the  species  of 
animal  to  be  used  depends  in  part  on  its  demonstrated  ability  to 
produce  antitoxin  and  in  part  in  commercial  establishments  on  the 
possibility  of  obtaining  large  volumes  of  immune  serum.  It  is  often 
found  desirable  to  select  a  species  which  has  natural  immunity  to 
certain  toxins  and  by  inoculation  raise  that  immunity  to  a  higher 
degree.  This  may  be  accomplished  with  less  difficulty  than  if  a 
susceptible  species  were  used.  This  is  true  of  the  use  of  the  horse 
in  producing  gas  bacillus  antitoxin.  The  same  principle  is  employed  by 
Kyes  in  using  fowl  for  the  production  of  an  anti-pneumococcus 
serum,  although  in  this  case  it  is  not  clear  that  the  serum  is  an  anti- 
toxic serum.  Kyes  states  that  the  antiserum  is  antibacterial,  i.e., 
agglutinating  and  bacteriolytic.  Especially  in  the  case  of  suscep- 
tible animals  and  also  in  relatively  immune  animals  it  may  be  neces- 
sary either  to  dilute  the  toxin  to  a  very  high  degree  or  to  attenuate 
it  by  other  means,  and  thus  consume  considerable  time  in  develop- 
ing a  high  degree  of  immunity.  For  immunizing  guinea-pigs  against 
diphtheria  toxin  Behring  and  Kitasato  used  iodine  terchloride, 
and  Roux  and  Martin,  Lugol's  solution.  Frankel  heated  the 
toxin  to  60  degrees.  Behring  in  the  case  of  tetanus  toxin  used 
a  neutralized  mixture  of  toxin  and  antitoxin,  gradually  reduc- 
ing the  amount  of  antitoxin,  and  finally  using  unmodified  toxin.  In 
a  sense  this  latter  method  has  been  employed  by  Behring  and  by 
Park  for  producing  active  immunity  to  diphtheria  in  children,  al- 
though here  it  has  been  found  unnecessary  to  use  pure  toxin  with- 
out antitoxin  to  attain  the  desired  result.  It  is  believed  that  after 
injection  there  is  a  dissociation  of  the  mixture,  which  permits  the 


42  THE  PRINCIPLES  OF  IMMUNOLOGY 

toxin  to  induce  active  antitoxin  production  by  the  patient's  own 
body.  In  the  work  with  animals  the  injections  are  given  at  inter- 
vals of  a  few  days,  sometimes  interspersed  with  rest  periods  of 
about  a  week,  until  testing  of  small  amounts  of  serum  shows  that  a 
satisfactory  result  has  been  attained. 

Technic  of  Producing  Diphtheria  Toxin,  and  Antitoxin. — Some  details  of 
the  technic  of  producing  diphtheria  antitoxin  may  well  serve  as  an  example  of  the 
general  phases  of  the  method.  It  was  early  noted  that  different  strains  of  the 
diphtheria  bacillus  produced  toxins  of  variable  strength  and  also  that  the  same 
strain  showed  slight  variation.  Finally  the  strain  isolated  by  Park  and  Williams, 
now  well  known  as  Park  No.  8,  a  strong  toxin  producer,  was  selected  as  a  standard 
and  is  so  used  throughout  the  world.  In  order  to  obtain  the  best  aerobic  conditions, 
a  wide-bottom  flask  is  employed  so  as  to  expose  a  large  surface  of  the  medium, 
"bob"  veal  broth  being  selected  as  most  desirable.  The  culture  is  planted  super- 
ficially and  allowed  to  grow  for  seven  or  eight  days.  It  is  wise  to  use  a  culture  that 
has  been  grown  for  several  generations  on  the  surface  of  broth  tubes,  the  tubes 
so  inclined  as  to  expose  a  large  broth  surface.  This  accustoms  the  organisms  to 
freely  aerobic  conditions.  After  the  period  of  growth  in  the  flask  the  organisms 
are  killed  by  formaldehyde  or  by  phenol,  or  even  by  heat,  and  the  broth  filtered 
either  through  paper  or  through  a  porcelain  filter,  preserved  with  toluol  and  per- 
mitted to  "  ripen."  This  ripening  is  made  desirable  because  of  progressive  deteriora- 
tion of  the  toxin,  a  phenomenon  which  will  be  more  profitably  taken  up  in  the 
discussion  of  the  toxin-antitoxin  combination. 

The  following  table  shows  a  scheme  as  actually  practised  for  producing 
diphtheria  antitoxin.  All  the  injections  are  subcutaneous. 

INJECTION  SCHEME  FOR  PRODUCTION  OF  DIPHTHERIA  ANTITOXIN 
July    22      6,000  units  antitoxin 

25  400  minimum  lethal  doses  of  toxin  (see  page  45) 
27        800  minimum  lethal  doses  of  toxin 

29  1,200  minimum  lethal  doses  of  toxin 

Aug.  i  i, 600  minimum  lethal  doses  of  toxin 

3  2,000  minimum  lethal  doses  of  toxin 

5  2,500  minimum  lethal  doses  of  toxin 

8  3,000  minimum  lethal  doses  of  toxin 

10  3,600  minimum  lethal  doses  of  toxin 

12  4,400  minimum  lethal  doses  of  toxin 

15  5,200  minimum  lethal  doses  of  toxin 

17  6,200  minimum  lethal  doses  of  toxin 

19  7,200  minimum  lethal  doses  of  toxin 

22  8,500  minimum  lethal  doses  of  toxin 

24  10,000  minimum  lethal  doses  of  toxin 

26  13,000  minimum  lethal  doses  of  toxin 
29  16,000  minimum  lethal  doses  of  toxin 
31  20,000  minimum  lethal  doses  of  toxin 

Sept.    2   24,000 


5  28,000 

7  32,000 

9  36,000 

12  40,000 


Lengthen  intervals  by  24  hours  if  made 
necessary  by  severe  reaction. 


Trial  bleeding  separation  of  and  testing  of  serum,  September  21.  After  that 
if  further  immunization  is  necessary  the  dose  is  raised  5000  M.L.D.  each  injection. 

The  Nature  of  Antitoxins. — The  serum  thus  obtained  contains 
the  antitoxin.  The  exact  nature  of  the  antitoxin  is  unknown,  but 
chemical  examination  and  other  studies  have  thrown  a  certain 
amount  of  light  upon  its  properties.  If  we  can  accept  the  division 
of  the  serum  protein  into  fibrinogen,  euglobulin  and  pseudo-globulin 
by  precipitation  with  magnesium  sulphate  or  ammonium  sulphate, 
the  antitoxin  is  found  in  that  water-soluble  fraction  known  as 


TOXINS  AND  ANTITOXINS 


43 


pseudo-globulin,  which  constitutes  about  78  per  cent,  of  the  serum 
protein.  This  fact  is  taken  advantage  of  in  the  so-called  concentra- 
tion of  antitoxin,  in  which  the  pseudo-globulin  is  thrown  down  by 
the  addition  of  ammonium  sulphate.  Heinemann  states  that  pseudo- 
globulins  may  be  broken  into  fractions,  one  of  which  contains  the 
antitoxin  in  highly  concentrated 
form,  thus  making  the  bulk  even 
smaller  than  by  the  use  of  pseudo- 
globulin.  The  precipitate  is  col- 
lected, dialyzed  free  of  salt,  and 
taken  up  in  water,  the  final  volume 
being  considerably  less  than  the 
original  amount  of  serum,  there- 
fore containing  a  greater  number 
of  antitoxic  units  per  c.c.  than  the 
whole  serum.  This  does  not  mean 
that  the  antitoxin  is  necessarily  a 
globulin,  for  it  resists  trypsin  diges- 
tion in  greater  degree  than  does 
globulin.  It  is,  however,  an  elec- 
tro-positive colloid.  Antitoxin  is 
not  thrown  down  in  indifferent 
precipitates,  and  in  this  respect  dif- 
fers from  the  enzymes,  nor  does  it 
operate  in  the  same  quantitative  re- 
lations as  enzymes.  The  large  size 
of  the  antitoxin  molecule  is  indi- 
cated by  the  famous  Martin  and 
Cherry  experiment,  which  showed 
that  if  toxin  and  antitoxin  are 
mixed  and  passed  through  gelatin 

filters  the  toxin  appears  first.      The    PIG.   i.— Apparatus  for  filtration  through  porce- 
Same     point     Was     brought     OUt     by    ^in  of  small  quantities  of  material.     ] 

Arrhenius   and    M  a  d  s  e  n  ,    who 
showed  that  toxin  diffuses  ten  times 
more  rapidly  than  antitoxin.    Anti- 
toxin is  injured  by  moist  heat  of  60°  to  70°  C,  destroyed  by  moist  heat 
of  100°  C.,  and  by  dry  heat  of  140°  C. 

The  influence  of  temperature  on  antitoxin  is  of  the  utmost  prac- 
tical importance  in  regard  to  its  preservation  for  therapeusis. 
Anderson  has  estimated  the  yearly  deterioration  at  different  tem- 
peratures as  follows : 


rubber  tube  and  the  suction  apparatus  must  be 
inserted  a  small  trap  to  prevent  entrance  into  the 
flask  of  water  when  suction  is  released.  The  trap 
is  a  salt  neck  bottle  with  an  inlet  and  outlet  tube 
through  a  rubber  stopper. 


Temperature 


5°C. 


Yearly  deterioration 
20  per  cent. 
10  per  cent. 
6  per  cent. 


44  THE  PRINCIPLES  OF  IMMUNOLOGY 

McConkey  has  given  the  following  rates  of  deterioration : 

Temperature  Deterioration  in  6  months 

36°  C.  37  per  cent. 

6°-i6°  C.  14  per  cent. 

Ice  chest  7  per  cent. 

The  second  figures  in  McConkey's  table  indicate  room  temper- 
ature in  winter  and  summer.  Although  the  two  series  of  investi- 
gations differ  in  actual  figures,  they  serve  to  show  that  the  only 
temperatures  for  satisfactory  preservation  are  those  of  the  ice  chest. 
Antitoxin  is  destroyed  by  putrefaction  of  the  serum,  by  acids  and 
alkalies,  by  ultra-violet  rays  and  deteriorates  in  solution,  by  expo- 
sure to  light  and  air.  Ingestion  into  the  alimentary  tract  destroys 
antitoxin.  Nevertheless,  it  is  stated  that  suckling  infants  can  absorb 
antitoxin  from  the  mother's  milk.  Toxin  disappears  from  the  blood 
in  the  neighborhood  of  from  seven  to  eleven  days  after  injection,  it 
being  in  part  destroyed,  in  part  bound  by  the  tissues,  and  in  very 
small  part  excreted  in  the  urine.  Antitoxin  appears  in  man  very 
early  in  life,  as  determined  by  the  Schick  test  (see  page  53).  It  has 
not  been  proven  why  the  antitoxin  develops,  that  is,  whether  it  is 
natural  or  the  result  of  slight  attacks  of  the  disease.  As  indicated 
above,  it  may  possibly  be  transferred  in  mother's  milk.  Sherman 
states  that  lysins  and  complement  are  inappreciable  in  the  youngest 
swine  embryos,  but  that  after  the  ninth  week  of  gestation  they  can 
be  demonstrated  in  varying  amount.  Whether  they  are  autoch- 
thonous or  transmitted  from  the  mother  has  not  been  determined. 
Wells  states  that,  "  taken  together,  the  evidence  indicates  a  closer 
resemblance  of  antitoxins  to  proteins  than  has  been  shown  for  the 
toxins,  and  all  attempts  to  separate  antitoxins  from  proteins  have 
so  far  failed." 

The  manner  in  which  antitoxin  neutralizes  toxin  is  the  subject 
of  much  discussion,  experiment,  and  hypothesis.  Before  discussing 
the  matter  from  a  theoretical  point  of  view,  it  is  advisable  to  explain 
some  of  the  technical  operations  in  the  standardization  or  titration  of 
the  antitoxin.  From  the  practical  point  of  view  this  is  now  rela- 
tively simple,  although  requiring  an  extremely  precise  method,  but 
the  earlier  investigators  were  beset  with  many  difficulties. 

Standardization  of  Diphtheria  Antitoxin. — In  diseases  such  as 
diphtheria  and  tetanus,  where  the  symptoms  are  the  results  of  the 
action  of  the  toxin,  it  is  necessary  to  determine  the  amount  of  anti- 
toxin required  to  protect  an  animal  against  the  effect  of  a  given 
amount  of  toxin.  The  earlier  investigators  attempted  to  determine 
the  amount  of  antitoxic  serum  necessary  to  protect  against  inocula- 
tions with  living  organisms,  but  the  variability  in  biological  proper- 
ties of  growth  and  toxin  production,  infection,  and  resistance,  soon 
showed  the  unreliability  of  this  method.  Behring  then  took  up  the 
determination  of  antitoxin  against  toxin,  but  found  it  difficult  to 
standardize  such  a  method  over  a  wide  geographic  area  because  of 


TOXINS  AND  ANTITOXINS  45 

differences  in  bacterial  strains  and  variations  in  the  same  strains 
growing  under  even  slightly  different  conditions.  Ehrlich  sought  to 
reduce  the  factor  of  error  by  determining  the  antitoxic  "  unit "  as 
the  amount  of  antitoxic  serum  necessary  to  protect  against  ten  times 
the  minimum  lethal  dose.  Even  this  was  unsatisfactory,  and  Behring 
and  Ehrlich  in  collaboration  settled  upon  an  arbitrary  method  of 
determining  a  "  normal "  toxin  and  a  "  normal  "  therapeutic  serum. 
The  "normal"  toxin  contained  in  i.o  c.c.  one  hundred  times  the 
minimum  lethal  dose  for  a  guinea-pig  of  250  grams.  The  "  normal  " 
therapeutic  serum  was  tested  and  diluted  so  that  o.i  c.c.  contained 
sufficient  antitoxin  to  neutralize  i.o  c.c.  of  the  "normal"  toxin  or, 
in  other  words,  i.o  c.c.  antitoxic  serum,  as  a  unit,  was  capable  of 
neutralizing  100  minimum  lethal  doses  of  toxin.  The  fundamental 
error,  however,  had  not  been  overcome  by  this  method,  and  it  was 
found  that  no  method  which  had  as  its  basis  a  toxin,  could  be  ap- 
plied over  a  large  area  and  the  method  was  finally  abandoned. 
The  toxin  deteriorates  rapidly  on  standing,  and  even  though 
after  a  time  it  becomes  fairly  stable,  it  still  is  insufficiently  so 
to  justify  its  use  for  purposes  of  standardization.  On  the  other 
hand,  the  antitoxic  serum  resists  drying  for  an  indefinite  period,  and 
if  used  as  a  standard  can  be  shipped  great  distances.  The  standard 
in  this  country  has  been  established  by  the  United  States  Public 
Health  Service.  The  unit  of  antitoxin  as  now  used  has  no  direct 
relation  to  the  unit  of  "  normal "  therapeutic  serum  as  defined  by 
Behring  and  Ehrlich,  but  by  interchange  between  nations  it  is 
practically  constant  throughout  the  world.  Hence,  if  a  laboratory 
wishes  to  prepare  an  antitoxin  the  standard  unit  of  antitoxin  can  be 
obtained  from  the  Public  Health  Service.  With  the  standard  anti- 
toxin on  hand,  the  antitoxic  content  of  a  newly  prepared  antiserum 
may  be  determined.  This  must  be  done  through  the  medium  of  a 
toxin  whose  strength  is  titrated  against  the  standard  antitoxin ;  the 
toxin  is  thereby  standardized,  so  that  the  strength  of  the  new  anti- 
toxic serum  can  be  measured.  The  toxin  must  be  one  which  has 
been  ripened,  so  that  any  deterioration  during  the  few  days'  time 
necessary  for  titration  against  the  standard  immune  serum  and  then 
against  the  new  serum,  is  reduced  to  a  negligible  minimum.  In 
order  to  make  the  titration  against  the  standard  antitoxic  unit  some- 
what easier  it  is  well  to  know  the  minimum  lethal  dose  of  toxin. 
There  are  then  to  be  determined : 

1.  The  minimum  lethal  dose  of  toxin  (M.L.D.). 

2.  The  L0  dose  of  antitoxin. 

3.  The  L+  dose  of  antitoxin. 

The  minimum  lethal  dose  of  toxin  is  determined  by  injecting  subcuta- 
neously,  varying  doses  of  toxin  into  a  series  of  guinea-pigs  250  grams  in 
weight.  Healthy  pigs  of  this  weight  are  usually  young  and  less  expensive 
than  fully-grown  animals.  The  M.L.D.  is  the  smallest  dose  that  kills  a 
pig  in  from  four  to  five  days.  Less  than  four  days  means  too  great  strength, 


46  THE  PRINCIPLES  OF  IMMUNOLOGY 

more  than  five  days  too  little  strength.  It  can  be  seen  that  the  selection  of 
the  weight  of  the  pigs  and  the  length  of  time  are  arbitrary  but  universal 
standards.  A  strong  toxin  might  give  results  as  follows: 

Guinea-pig  Toxin  dose  Result 

1  0.0036  c.c.  Lives 

2  0.0038  c.c.  Dead  6  days 

3  0.0040  c.c.  Dead  4  days     8  hours 

4  0.0042  c.c.  Dead  3  days  20  hours 

5  0.0044  c.c.  Dead  2  days 

Guinea-pig  No.  3  died  at  the  right  time  interval  and  0.004  is  the  M.L.D. 
of  this  toxin.  Experiments  with  a  preliminary  series  using  more  widely  vary- 
ing doses  of  toxin  would  be  necessary  before  the  final  experiment  could 
be  set  up. 

The  Lo  dose  (Limes  null)  is  that  amount  of  toxin  which  is  so  thoroughly  satu- 
rated with  one  unit  of  antitoxin  that  neither  local  nor  general  symptoms 
appear  following  the  injection  of  the  mixture.  An  experiment  follows: 

Standard 
Guinea-pig      antitoxin  Toxin  Result 

1  i  unit  0.36  c.c.  No  reaction 

2  i  unit  0.38  c.c.  No  reaction 

3  i  unit  0.40  c.c.  Barely  visible  congestion 

4  i  unit  0.42  c.c.  Moderate  inflammation 

5  i  unit  0.44  c.c.  Distinct  inflammation 

In  this  experiment  the  dose  of  toxin,  0.40  c.c.,  given  pig  No.  3,  is  the  L0 
dose.  The  note  as  to  reaction  refers  to  the  shaven  site  of  injection. 

The  L+  dose  (Limes  death)  indicates  the  smallest  amount  of  toxin  which 
after  mixture  with  one  unit  of  antitoxin  will  produce  death  in  four-five  days. 
The  plus  sign  is  the  mark  used  in  English  texts  to  correspond  to  the  cross 
mark  used  in  German  literature  to  signify  death.  An  experiment  follows : 

Standard 
Guinea-pig  antitoxin  Toxin  Result 


unit  0.44  c.c.  Lives 

unit  0.46  c.c.  Dead  6  days 

unit  0.48  c.c.  Dead  4  days 

unit  0.50  c.c.  Dead  3  days 

unit  0.52  c.c.  Dead  2  days 


In  this  experiment  0.48  c.c.  given  guinea-pig  No.  3  is  the  L+  dose  of  toxin. 

Titration  of  Diphtheria  Antitoxin.— In  the  actual  titration  of  an 
antitoxin  as  practised  to-day  there  must  be  at  hand  a  standard  anti- 
toxin of  known  strength  as  well  as  a  toxin,  whose  M.L.D.  has  been 
at  least  approximately  determined.  The  antitoxin  has  been  dried 
in  a  vacuum  and  preserved  in  sealed  U-shaped  ampoules  which  con- 
tain the  antitoxin  in  one  arm  and  P2O5  or  some  other  hygroscopic 
substance  in  the  other  arm,  in  order  to  maintain  the  dryness  of  the 
antitoxin.  The  ampoule  is  best  kept  in  a  light-proof  box  in  the  re- 
frigerator. Against  this  antitoxin  the  L+  dose  of  a  toxin  is  deter- 
mined, and  against  this  toxin  the  new  antitoxin  is  titrated.  The 
amount  of  antitoxin  which  protects  against  the  L+  dose  for  four 
days  is  the  antitoxin  unit  of  the  new  serum.  In  preliminary  experi- 
ments the  antitoxin  is  roughly  titrated  in  dilutions  of  i :  100,  i :  200, 
1:300,  and  so  on.  In  each  case  the  antitoxin  is  used  in  i.o  c.c. 
amounts  and  the  toxin  so  diluted  that  2.0  c.c.  contain  the  M.L.D., 
the  two  being  mixed  and  allowed  to  stand  at  room  temperature  for 


TOXINS  AND  ANTITOXINS 


47 


one  hour.  The  Rosenau  glass  syringe  for  this  purpose  has  an 
oblique  side  arm  for  salt  solution,  so  that  after  the  toxin-antitoxin 
mixture  is  injected,  the  side  arm  is  swung  around,  emptying  the 
saline  into  the  main  body  of  the  syringe.  The  salt  solution  is  then 
injected,  thus  washing  out  the  remnants  of  the  toxin-antitoxin  mix- 
ture that  may  remain  in  the  lower  part  of  the  syringe  and  needle. 

The  following  experiment  will  serve  to  illustrate,  granting  that  the  pre- 
liminary titration  showed  a  strength  of  antitoxin  between  1—200  and  1-400. 


Antitoxin 

Toxin 

Guinea-pig    i  c.c.  of  each 
dilution 

2.0  C.C 

=  M.L.D. 

I 

-200 

2.O   C.C. 

2 

-220 

2.O   C.C. 

3 

-240 

2.0    C.C. 

4 

-260 

2.O    C.C. 

-280 

2.O    C.C. 

6 

-300 

2.O    C.C. 

7 

-320 

2.O    C.C. 

8 

-340 

2.O    C.C. 

9 

-360 

2.0    C.C. 

10 

-380 

2.0    C.C. 

Dead 
Dead 
Dead 
Dead 
Dead 
Dead 
Dead 
Dead 
Dead 
Dead 


Result 

5  days 
5  days 
4  days 
4  days 
3  days 
3  days 
3  days 
3  days 
2  days 
2  days 


Thus  doses  i  and  2  were  more  than  sufficient  to  protect  four  days,  and 
doses  5-10  were  insufficient.  Doses  3  and  4  protected  for  four  days,  and  in 
order  to  be  safe  dose  No.  3  of  1-240  would  be  selected.  If  the  antitoxic  unit  is 
1/240  of  i.o  c.c.,  each  c.c.  of  serum  contains  240  units  of  antitoxin.  In  com- 
mercial work,  the  practice  is  to  be  absolutely  on  the  safe  side,  and  the  next 
larger  dose  of  antitoxin  would  be  employed  as  the  unit,  and  the  serum 
marketed  as  containing  220  units  per  c.c. 

In  the  therapeutic  use  of  such  a  serum  the  unit 
content  of  the  serum  is  simply  a  guide  to  its  use, 
the  dose  employed  being  rather  on  an  empirical 
basis  than  otherwise,  because  of  the  uncertainty  of 
the  amount  of  toxin  present  in  the  body  of  the 
patient.  It  is  generally  assumed  that  the  larger 
the  extent  of  the  exudate,  the  greater  the  amount 
of  toxin  produced  and  the  larger  the  absorbing  sur- 
face, but  it  can  readily  be  seen  from  the  theoretical 
standpoint  that  variations  may  be  produced  by  dif- 
ferences in  toxin  production  by  the  different  strains 
of  bacillus  diphtheriae  which  may  be  encountered 
in  patients.  It  is  unwise  to  stress  this  latter  possi- 
bility and  preferable  to  regulate  the  dosage  on  the 
former  basis.  More  will  be  said  later  regarding 
therapeusis. 

The  Toxin-antitoxin  Union. — T  he  E  hrlic  h 
Theory. — With  the  foregoing  practical  considera- 
tion of  antitoxin  titration  in  mind,  the  theoretical 
problems  of  the  nature  of  the  toxin-antitoxin  com- 
bination  will  be  taken  up.  The  simplest  conception 
is  that  antitoxin  neutralizes  toxin  in  the  same  way 
that  a  strong  acid  neutralizes  a  strong  base.  As  has  been  seen, 
the  neutralization  is  quantitative  and  follows  in  a  general  way 


48  THE  PRINCIPLES  OF  IMMUNOLOGY 

the  law  of  multiple  proportions.  If  this  were  true,  how- 
ever, the  L+  dose  which  in  combination  with  the  antitoxin  unit 
kills  a  pig  in  four  days  should  contain  one  unit  more  of  toxin 
(i  M.L.D.)  than  the  L0  dose  which  just  fails  to  produce  symptoms. 
Reference  to  the  experiments  offered  to  illustrate  the  determination 
of  M.L.D. ,  L0  dose,  and  L+  dose  show  that  the  M.L.D.  of  the  toxin 
was  0.004  c-c-» tne  L0  dose  of  toxin  was  0.40  c.c.  or  one  hundred  times 
the  M.L.D.,  and  the  L+  dose  0.48  c.c.  or  one  hundred  and  twenty 
times  the  M.L.D.  The  difference  between  L0  and  L+  doses  is,  there- 
fore, twenty  times  the  M.L.D.  instead  of  exactly  equal  to  it.  This 
has  been  interpreted  to  indicate  that  some  body  or  bodies,  other  than 
the  toxin,  has  combined  with  the  antitoxin,  thus  limiting  its  ability 
to  combine  with  toxin.  Ehrlich,  after  numerous  experiments  and 
hypotheses,  reached  the  assumption  that  the  toxic  broth  contains 
two  bodies  other  than  toxin,  which  he  named  toxon  and  toxoid.  The 
toxon  is  a  body  with  a  smaller  degree  of  affinity  for  the  antitoxin 
than  has  the  toxin.  In  a  determination  of  the  L0  dose  the  antitoxin 
neutralizes  both  toxin  and  toxon,  so  that  no  symptoms  appear,  but 
if  more  toxin  be  added  to  the  mixture  it  combines  with  antitoxin, 
displacing  the  more  loosely  combined  toxon.  Finally,  after  suffi- 
cient addition  of  toxin  the  antitoxin  is  fully  saturated,  and  any  addi- 
tional toxin  will  be  free,  and  if  in  sufficient  quantity  (i  M.L.D.) 
will  lead  to  the  death  of  the  experimental  animal.  In  more  detail 
the  20  M.L.D.'s  necessary  to  make  the  difference  between  the  L0  and 
L+  doses  were  so  used  that  19  M.L.D.'s  were  employed  to  displace  a 
proportionate  amount  of  toxon  and  toxoid  from  combination  with 
the  antitoxin  unit,  and  the  remaining  i  M.L.D.  sufficed  to  kill  the 
pig  in  four  days.  If  more  than  i  M.L.D.  were  present  in  excess 
death  would  ensue  after  a  shorter  period,  and  if  less  than  i  M.L.D. 
were  present  death  would  occur  later  than  four  days  or  not  at  all. 
It  is  believed,  on  the  basis  both  of  experimental  and  clinical  obser- 
vation, that  toxon  is  responsible  for  the  late  paralyses  of  diphtheria. 
The  conception  of  the  toxoid  is  based  on  the  Ehrlich  assumption 
that  the  toxin  molecule  has  a  toxic  fraction  or  "  toxophore  group  " 
and  a  combining  fraction  or  "  haptophore  group."  A  toxin  will 
retain  its  binding  power  for  antitoxin  for  a  considerable  length  of 
time  with  little  change  in  the  L+  dose,  but  with  marked  deterioration 
of  toxic  power  and  corresponding  reduction  of  the  M.L.D.  This  is 
interpreted  as  meaning  that  the  toxic  fraction  is  labile  and  the  com- 
bining fraction  much  more  stable.  The  toxoid,  then,  is  the  toxin 
molecule  so  altered  that  its  toxic  part  is  reduced  and  the  combining 
part  practically  intact.  As  can  readily  be  seen,  this  can  account 
also  in  part  for  the  discrepancy  between  L0  and  L+  dose.  The 
discrepancy  between  L0  and  L+  dose  in  fresh  toxic  broth  is  be- 
lieved to  be  due  to  the  presence  of  toxon  rather  than  toxoid,  be- 
cause too  short  a  time  has  elapsed  to  account  for  toxoid  formation. 
As  the  toxic  broth  becomes  older  the  discrepancy  becomes  greater, 
even  after  a  relative  equilibrium  has  been  established,  and  the  differ- 


TOXINS  AND  ANTITOXINS  49 

ence  is  believed  to  be  due  to  the  progressive  formation  of  toxoid 
from  toxin  and  perhaps  also  from  toxon.  Ehrlich  and  also  Madsen 
found  that  the  combination  of  one  antitoxic  unit  with  toxin  in  the 
determination  of  the  L0  dose  was  in  multiples  of  100  M.L.D/s. 
These  multiples  were  rarely  less  than  100  and  never  more  than  200. 
This  would  indicate  that  the  multiple  is  not  less  than  100,  but  even 
though  values  of  200  are  not  obtainable,  the  failure  may  be  ex- 
plained by  the  fact  that  pure  toxin  is  not  procurable.  By  means  of 
the  phenomenon  of  "  partial  absorption "  Ehrlich  established  a 
formula  for  the  antitoxin-toxin  combination  which  he  expressed  as 
"toxin200  antitoxin."  This  has  been  illustrated  by  means  of  a  toxin- 
antitoxin  "  spectrum  "  based  on  a  total  valency  of  200,  the  total  valency 
including  toxin,  protoxoid,  and  toxon.  In  spite  of  the  great  academic 
interest  of  this  discussion,  its  immediate  practical  value  is  not  apparent 
and  the  reader  is  referred  to  larger  works  for  complete  discussion. 

Objections  to  the  Ehrlich  Theory. — As  indicated  previously,  the 
Ehrlich  hypothesis  is  based  on  the  assumption  that  the  toxin-anti- 
toxin reaction  follows  in  a  fairly  close  manner  the  chemical  reaction 
between  a  strong  acid  and  a  strong  base.  Certain  features  of  the 
process  of  combination  support  the  idea  of  chemical  union,  as,  for 
example,  the  fact  that  warmth  accelerates  the  reaction,  dilution 
slows  it.  Furthermore,  there  is  a  liberation  of  heat  in  the  reac- 
tion, that  is  to  say,  about  half  as  much  heat  per  gram  molecule  as 
would  be  liberated  by  the  reaction  between  a  strong  acid  and  a 
strong  base.  It  is  well  known,  however,  that  the  union  of  toxin  and 
antitoxin  is  loose  and  within  certain  limits  reversible.  The  Martin 
and  Cherry  experiment  referred  to  earlier  in  this  chapter  is  of  great 
importance  in  this  connection.  They  mixed  snake  venom  and  anti- 
toxin to  a  point  of  neutralization  and  filtered  through  gelatin  filters, 
with  the  result  that  the  toxin  came  through  the  filter  first.  This 
they  interpreted  as  being  due  to  the  smaller  size  of  the  toxin  mole- 
cule. It  also  shows  the  looseness  of  combination  and  the  reversi- 
bility of  the  reaction.  They  further  showed  that  the  longer  the 
mixture  stands,  the  smaller  the  amount  of  toxin  that  comes 
through  the  filter.  Zinsser  states  that  the  "  chief  value  of  these 
experiments  lies  in  their  proof  of  the  element  of  time  as  an 
important  factor  in  the  toxin-antitoxin  union."  Calmette  had  previ- 
ously shown  that  venoms  of  certain  snakes  would  remain  virulent 
after  heating  even  to  100  degrees,  and  that  the  antitoxins  were 
thermolabile.  He  demonstrated  that  if  the  two  were  mixed  so  as  to 
be  non-toxic,  subsequent  heating  would  liberate  the  toxin  probably 
through  thermic  destruction  of  the  antitoxin.  If  the  union  were  a 
fixed  one,  this  should  not  have  been  true.  Martin  and  Cherry  failed 
to  confirm  this  with  the  venom  of  an  Australian  snake,  but  this  can- 
not be  regarded  as  a  refutation  of  Calmette's  work,  especially  as  the 
principle  was  found  to  apply  to  other  toxins  and  antitoxins. 
Morgenroth  showed  that  acidulation  with  HC1  of  a  venom  lysin- 
antilysin  mixture  produced  an  acid-toxin  molecule  that  resisted 
4 


50  THE  PRINCIPLES  OF  IMMUNOLOGY 

heat  and  could  by  heat  be  dissociated  from  the  thermolabile  anti- 
lysin.  The  subsequent  chemical  neutralization  of  the  toxin  (or 
lysin)  resulted  in  the  restoration  of  its  toxicity.  In  this  laboratory 
Wahl  has  shown  that  titration  of  diphtheria  toxin  using  normal 
guinea-pigs  in  one  series  and  guinea-pigs  with  only  one  kidney  in 
another,  gives  materially  different  results.  These  experiments  were 
carefully  controlled  and  may  be  offered  as  a  further  indication  of  the 
loose  combination  and  its  corollary  the  reversibility  of  the  reaction, 
on  the  probable  assumption  that  the  toxin  is  more  readily  excreted 
by  the  animals  with  two  kidneys  than  by  those  with  one.  The 
recitation  of  these  few  experiments  to  which  others  might  be  added 
is  sufficient  to  illustrate  the  objection  to  the  Ehrlich  theory  of  fixed 
combination,  and  two  other  important  hypotheses  are  offered  for 
consideration  :  (i)  The  conception  that  the  combination  follows  the 
law  of  mass  action  and  (2)  the  theory  of  colloidal  reaction. 

The  Law  of  Mass  Action. — The  application  of  the  law  of  mass 
action  has  been  worked  on  by  Arrhenius  and  Madsen  principally. 
This  law  is  usually  illustrated  in  the  chemical  laboratories  by  the 
reaction  between  one  gram  molecule  ethyl  alcohol  and  one  gram 
molecule  acetic  acid  which  yields  ethyl  acetate  and  water,  the  reac- 
tion, however,  stopping  at  a  point  of  equilibrium  where  there  is 
found  in  the  mixture  ^3  gram  molecule  alcohol,  l/$  gram  molecule 
acetic  acid,  2/z  gram  molecule  ethyl  acetate,  and  2/z  gram  molecule 
water.  The  same  end-result  is  obtained  if  instead  of  mixing  ethyl 
alcohol  and  acetic  acid,  we  mix  ethyl  acetate  and  water,  thus  indicat- 
ing the  reversibility  of  the  reaction  as  stated  in  the  formula : 

C,H6OH  +  CH3COOH  ^±  CH8COOC2H5  +  H2O 

Arrhenius  and  Madsen  compared  the  reaction  between  tetanoly- 
sin  and  its  antitoxin  and  the  reaction  between  boric  acid  and  am- 
monia. This  was  of  advantage  because  ammonia  is  hemolytic  and 
boric  acid  is  not.  Thus  a  reversible  reaction  is  found  in  which  the 
addition  of  boric  acid  reduces  the  hemolytic  activity  of  the  ammonia. 
As  with  the  alcohol-acetic  acid  experiment,  however,  a  point  of 
equilibrium  is  established  whereby  there  .always  remains  a  small 
amount  of  free  ammonia  in  spite  of  the  addition  of  boric  acid  to  a 
point  of  saturation.  The  same  general  proposition  holds  in  regard 
to  tetanolysin  and  antilysin,  and  these  authors  were  able  to  con- 
struct similar  curves  of  neutralization  for  both  of  these  reactions. 
With  this  idea  as  a  basis,  the  late  paralyses  of  diphtheria,  either  ex- 
perimental or  clinical,  would  depend  not  upon  the  toxon  of  Ehrlich, 
but  rather  upon  a  small  non-fatal  amount  of  toxin  that  is  never  com- 
pletely neutralized  in  the  reaction. 

The  Danysz  Effect. — It  will  readily  be  seen,  however,  that  the 
reversible  reactions  illustrating  the  law  of  mass  action  deal  with 
crystalloids,  while  it  is  probable  both  toxins  and  antitoxins  are  of 
colloidal  character.  Certain  colloids  are  known  as  "  reversible  col- 


TOXINS  AND  ANTITOXINS  51 

loids,"  but  as  yet  there  is  little  definite  proof  that  reversible  reac- 
tions between  two  colloids  take  place.  According  to  certain  inter- 
pretations, the  most  important  observation  in  support  of  the 
colloidal  theory  is  the  Danysz  effect.  If  the  toxin  is  added  to  the 
antitoxin  in  fractions  with  an  interval  of  time  elapsing  between,  less 
toxin  is  needed  to  saturate  the  antitoxin  than  if  the  toxin  were  added 
in  one  volume.  In  other  words,  if  i.o  c.c.  toxin  were  saturated 
in  the  usual  way  with  o.i  c.c.  antitoxin  and  if  in  another  test-tube 
the  toxin  is  added  to  the  same  amount  of  antitoxin,  not  in  a  single 
dose,  but  in  successive  doses  of  0.2  c.c.,  until  i.o  c.c.  is  present,  this 
latter  mixture  instead  of  being  neutral  would  be  toxic.  Wells,  how- 
ever, states  that  this  "  indicates  that  the  toxin  antitoxin  union  is 
physical  rather  than  chemical,  for  it  seems  to  be  quite  analogous  to 
such  a  phenomenon  as  the  taking  up  of  more  dye  by  several  pieces 
of  blotting  paper  added  in  series  to  a  dye  solution,  than  by  the  same 
amount  of  paper  added  in  one  piece."  Of  somewhat  similar  import 
is  the  absorption  theory  of  Bordet  and  of  Landsteiner,  which  states 
that  when  toxin  is  added  to  antitoxin  in  smaller  quantities  than 
saturation,  let  us  say  five  molecules  of  antitoxin  to  ten  of  toxin,  this 
does  not  result  in  complete  molecular  combination  with  five  mole- 
cules of  toxin,  but  rather  in  half  saturation  of  the  entire  ten  mole- 
cules of  toxin.  This  results  in  attenuation  of  the  toxin,  so  that 
instead  of  there  being  five  free  molecules  of  toxin  there  are  ten  units  of 
partly  detoxified  toxin.  It  is  not  to  be  expected  that  this  follows  in  exact 
arithmetical  progression,  but  Biltz  has  made  comparisons  with  absorp- 
tion phenomena  in  general  and  finds  fairly  consistent  results. 

In  summary  it  may  be  said  that  in  explaining  the  union  of  toxin  and 
antitoxin  the  Ehrlich  hypothesis  does  not  withstand  critical  examination 
and  that  the  reaction  is  in  all  likelihood  of  an  intricate  physico-chemical 
nature  referable,  in  part  at  least,  to  the  probable  colloidal  nature  of  the 
reacting  bodies,  but  not  as  yet  satisfactorily  explained. 

Therapeutic  Use  of  Diphtheria  Antitoxin. — In  1892  von  Behring 
and  Wernicke  found  that  the  serum  of  animals  immunized  against 
diphtheria  toxin  protects  other  animals  of  the  same  and  different 
species  against  the  action  of  the  toxin.  In  1894  Roux  demonstrated 
the  value  of  the  treatment  of  diphtheria  in  man  by  means  of  anti- 
toxic serum.  This  method  of  treatment  rapidly  attained  widespread 
use  and  has  markedly  reduced  the  mortality  from  the  disease. 
Numerous  statistical  studies  have  been  made  since  that  time,  and 
it  is  safe  to  say  that  the  introduction  of  antitoxin  treatment  has 
reduced  mortality  from  approximately  40  per  cent,  to  approximately 
7  per  cent.  In  interpreting  the  figures  it  has  been  found  necessary 
to  take  account  of  two  important  factors;  namely,  the  cases  of 
laryngeal  diphtheria  and  the  time  at  which  treatment  is  instituted. 
Laryngeal  diphtheria  presents  not  only  the  element  of  toxic  absorp- 
tion, but  in  addition  mechanical  obstruction  to  respiration  and 
the  possibility  of  extension  downward,  so  as  to  produce  pneumonia. 
Furthermore,  the  operative  procedures  for  relieving  the  respiratory 


THE  PRINCIPLES  OF  IMMUNOLOGY 


obstruction  are  such  as  to  introduce  an  additional  minor  element  of 
danger.  The  accidents  following  tracheotomy  are  distinctly  more 
numerous  than  those  following  intubation,  but  neither  operation  can 
be  regarded  as  absolutely  without  risk.  It  is  now  well  established 
that  the  earlier  in  the  course  of  disease  the  antitoxin  is  administered 
the  more  favorable  is  the  prognosis.  In  order  to  present  this 
graphically,  however,  we  insert  the  following  table  taken  in  large 
part  from  Dieudonne  and  Weichardt : 


Total 
number 
of  cases 

Mortal- 
ity per- 
centage 

ISt 

day 

2nd 
day 

3rd 
day 

4th 
day 

5th 
day 

6th 

day 

After 
6th 
day 

Welch 

I48Q 

14.2 

2.^ 

8.1 

13.5 

19.0 

29.3 

34.1 

33.7 

Hilbert 

**rwif 

2428 

T^ 

18.3 

•o 
2.2 

7.6 

I7.I 

23.8 

33.9 

34.1 

38.2 

American    Pediatric 

^T^ 

fcV^»O 

Society   

S794 

12.3 

4-99 

7.4 

8.8 

2O.7 

35.3 

Brook    Hospital, 

\}  /  "T^ 

London 

8007 

Q.5 

o.o 

A.  3 

II.  12 

17.24 

18.72 

Germany 

71 

7*C/ 

*T*O 

*  /  "-^T- 

Kais-Gesundh.  Amb- 

tes  (Berlin)  (Sam- 

melforschung)  .... 

958i 

15-5 

6.6 

8-3 

12.9 

17.0 

23.2 

26.9 

Russia 

(Rauchfusz)     (Sam- 

melforschung)  

44,631 

14.6 

3-7 

8.2 

16.2 

25.9 

Austria 

Sammelforschung 

(Sanitatswesen)  .  .  . 

1103 

12.6 

8.0 

6.6 

9.8 

25.5 

28.8 

30.7 

2I.O 

The  therapeutic  efficiency  of  the  antitoxin  also  varies  accord- 
ing to  the  method  of  administration.  According  to  Berghaus, 
intravenous  injections  are  five  hundred  times  more  effective  and 
intraperitoneal  are  eighty  to  ninety  times  more  effective  than  sub- 
cutaneous injections.  In  the  earlier  days  of  antitoxin  treatment  the 
method  was  almost  entirely  subcutaneous  injection,  but  subse- 
quently the  intravenous  method  was  employed  in  severely  toxic 
cases.  Injections  were  given  at  various  intervals,  usually  a  day 
apart,  until  the  disease  showed  marked  improvement.  The  studies 
of  Park  and  his  collaborators  have  modified  the  treatment  consid- 
erably. Park  was  able  to  show  that  a  single  dose  of  antitoxin  in 
sufficient  quantity  is  more  effective  in  neutralizing  the  circulating 
toxin  than  the  multiple  small  doses,  largely  because  of  the  fact  that 
with  subcutaneous  and  intramuscular  injections  the  absorption  is 
continuous,  whereas  during  the  period  usually  occupied  by  giving 
several  doses,  the  absorption  occurs  for  only  a  short  time  after  each 
injection.  Were  it  possible  to  determine  for  clinical  purposes  the  exact 
amount  of  toxin  absorption  during  the  disease  the  dosage  of  antitoxin 
could  be  accurately  regulated.  Unfortunately,  however,  different  strains 
of  the  bacilli  vary  in  capacity  for  production  of  toxin  and  the  depth 
and  extent  of  the  local  lesion,  as  well  as  the  nature  of  the  underlying 
tissues,  have  some  influence  upon  the  rate  and  amount  of  absorption. 


TOXINS  AND  ANTITOXINS  53 

Therefore,  the  actual  dose  employed  is  to  a  large  extent  upon  an  empiri- 
cal basis.  Park  recommends  the  following  table  of  doses  and  methods 
of  administration : 

DOSAGE  OF  UNITS  OF  ANTITOXIN  IN  DIPHTHERIA.    SINGLE  DOSE  ONLY. 

Infant,  ten  to  thirty  pounds  (under  two  years  of  age). 

Mild  Moderate  Severe  Malignant 

2,000  3,000  5,000  

3,000  5,000  10,000  10,000 

Child,  thirty  to  ninety  pounds  (under  fifteen  years  of  age). 
3,000  4,000  10,000  10,000 

4,000  10,000  15,000  20,000 

Adults,  ninety  pounds  and  over. 

3,000  5,ooo  10,000  15,000 

5,000  10,000  20,000  40,000 

METHOD  OF  ADMINISTRATION. 

Mild  Moderate  Severe  Malignant 

Subcutaneous     or    Intramuscular    or    Intramuscular    or    ^  intravenous  and 
intramuscular  subcutaneous  ^     intravenous       y2  intramuscular 

and  y2  intramus-        or  subcutaneous 
cular  or  subcu- 
taneous 

McCombie  recommends  the  following  dosages : 
Mild:    4000  to  8000  units  in  one  dose. 

Moderate:  12,000  to  16,000  units  in  one  dose  or  two  doses. 
Severe :  20,000  to  50,000  units  or  more  in  two  or  three  doses. 
Laryngeal:    16,000  to  24,000  as  initial  dose,  and  repeat  once  or  twice  according 
to  persistence  of  symptoms. 

Improvement  following  the  administration  of  antitoxin  is  strik- 
ing when  given  early  and  exhibits  itself  in  fall  of  temperature,  reduc- 
tion of  leucocytosis,  reduction  of  inflammation,  and  separation  of 
the  fibrinous  membrane.  This  improvement  varies  somewhat  with 
the  method  of  administration,  the  intravenous  method  effecting  im- 
provement in  somewhat  less  time  than  the  intramuscular,  and  the 
latter  in  somewhat  less  time  than  the  subcutaneous.  In  a  series  of 
cases  studied  in  the  City  Hospital  in  Cleveland  by  Ruh  the  intra- 
venous form  of  administration  was  followed  in  83  per  cent,  of  the 
cases  by  a  severe  general  reaction  with  chills  and  prostration  com- 
ing on  a  few  minutes  after  the  administration  and  lasting  for  about 
twenty  minutes.  At  the  present  time  it  is  impossible  to  state  the 
exact  cause  of  this  reaction.  Such  reaction  does  not  follow  subcu- 
taneous and  intramuscular  injections.  It  is  not  due  to  the  preserva- 
tive, nor  as  far  as  can  be  determined,  to  the  age  of  the  serum.  The 
most  reasonable  explanation  appears  to  us  to  be  that  the  reaction  is 
due  to  foreignness  of  the  horse  protein.  None  of  Ruh's  cases  showed 
prolonged  or  fatal  reactions,  and  it  is  not  probable  that  these  reac- 
tions represent  individual  hypersusceptibility,  because  if  this  were 
true  fatalities  would  be  likely  to  occur  (see  page  230). 

Natural  Immunity  to  Diphtheria — The  Schick  Test. — It  has  long 
been  known  that  many  individuals,  even  as  many  as  80  per  cent,  of 
adults  and  50  per  cent,  of  children,  are  immune  to  diphtheria,  as  indi- 


54  THE  PRINCIPLES  OF  IMMUNOLOGY 

cated  by  demonstrating  antitoxin  in  the  blood.  The  methods  of 
demonstration  were  not  easily  applicable  until  the  development 
of  the  Schick  test.  This  test  is  performed  by  injecting  intra- 
cutaneously  one-fiftieth  of  the  minimum  lethal  dose  of  a  specially 
prepared  toxin  contained  in  0.2  c.c.  of  salt  solution.  Injection  is  pref- 
erably on  the  flexor  surface  of  the  arm  or  forearm.  Six-day  broth 
cultures  of  the  organism  are  killed  with  phenol,  sedimented  in  the 
ice-box  for  two  or  three  days,  the  supernatant  fluid  filtered  through 
a  Berkefeld  candle,  and  the  clear  filtrate  accurately  standardized.  It 
is  well  to  keep  this  filtrate  for  several  months  or  a  year,  so  that  its 
rate  of  deterioration  is  reduced  to  a  minimum.  A  control  injection 
is  given  with  the  same  quantity  of  toxic  broth  heated  to  75°  C,  so 
as  to  destroy  the  toxin. 

The  reactions  to  injections  may  be  as  follows: 

A.  Positive  reaction.     This  indicates  that  no  antitoxin  is  present  in  the 
body,  thereby  permitting  the  toxin  to  act  upon  the  unprotected  cells.     Slight 
reaction  appears  in  from  twelve  to  twenty-four  hours  in  the  form  of  redness, 
which  becomes  more  distinct  in  from  twenty-four  to  forty-eight  hours,  reach- 
ing its  maximum  on  the  third  or  fourth  day,  then  gradually  disappearing  and 
leaving  an  area   of  scaling  and   brown   pigmentation.     The  area   attains   a 
diameter  of  10  to  20  mm.,  and  varies  in  intensity,  depending  on  the  sensitive- 
ness of  the  individual. 

B.  Negative  reaction.     If  no  distinct  reaction  appears  any  more  than  is 
seen  in  the  control  area  the  failure  to  react  indicates  that  an  amount  of  anti- 
toxin is  present  in  the  body  sufficient  to  neutralize  the  introduced  toxin.     Such 
a  reaction  in  a  child  of  about  three  years  of  age  probably  indicates  perma- 
nent immunity.     By  varying  the  quantity  of  toxin  injected,  the  amount  of 
antitoxin  can  be  titrated. 

C.  The  pseudo-reaction.  This  is  usually  urticaria]  in  nature,  appearing  some- 
times immediately  and  sometimes  in  from  six  to  eighteen  hours,  reaching  its 
maximum  on  the  third  or  fourth  day.     It  fails  to  leave  pigmentation  after  it 
subsides.     This    is   a   reaction   of   hypersusceptibility   to    the    protein    substances 
present  in  the  toxic  broth  as  the  result  of  the  autolysis  of  the  diphtheria 
bacilli,  and  is  in  nature  the  same  as  other  reactions  of  hypersusceptibility 
described  subsequently  (see  page  236).    Such  a  pseudo-reaction  may  intensify 
the  true  reaction  and  represent  a  summation  of  the  protein  reaction  and  a 
reaction  to  the  toxin.    This  must  be  taken  to  indicate  that  an  individual  may 
be  hypersensitive  to  the  proteins  of  diphtheria  bacilli  but  at  the  same  time 
not  possessing  in  his  circulating  blood  any  antitoxin.     The  differentiation 
depends  upon  the  difference  between  the   reaction  at  the  site  of  the  test 
injection  and  at  the  site  of  the  control  injection. 

Zingher  divides  the  positive  reactions  as  follows:  -f -f  indicates  a  strong 
positive  reaction  with  marked  local  redness,  infiltration,  and  occasionally  super- 
ficial vesiculation ;  -|-  indicates  positive  reaction  with  redness  but  little  or  no 
infiltration;  ±  indicates  moderately  positive  reaction  with  moderate  degree  of 
redness  and  no  local  infiltration ;  i  indicates  a  faintly  positive  reaction  with  only 
slight  redness  and  no  local  infiltration. 

The  test  has  been  found  to  be  of  great  value  in  determining  the 
immunity  of  groups  of  individuals,  particularly  in  institutions 
where  there  has  been  exposure  to  diphtheria.  It  has  also  given  con- 
siderable information  as  to  the  incidence  and  duration  of  this  variety 
of  active  immunity.  Immunity  to  diphtheria  may  be  derived  from 
the  mother  and  lasts  for  about  six  months  after  birth.  The  largest 
number  of  positives  is  found  from  the  ages  of  six  to  eighteen  months. 
This  gradually  decreases  throughout  life. 

Another  method  for  determining  the  presence  of  antitoxin  in  the 
blood  is  that  of  Romer.  This  depends  upon  the  well-known  fact 


PLATE  I. 


POSITIVE  SCHICK  REACTION 

Reaction  of  moderate  severity  seventy-two  hours  after  the 

intracutaneous  injection  of  one-fortieth  the  minimal  lethal 

dose  of  diphtheria  toxin.     Patient's  blood  serum  was  found  to 

contain  no  antitoxin  (International  Clinics). 


TOXINS  AND  ANTITOXINS  55 

that  the  intracutaneous  injection  of  toxin  into  guinea-pigs  leads  to 
localized  necrosis  in  the  course  of  forty-eight  hours.  The  minimum 
amount  of  toxin  sufficient  to  produce  necrosis  can  be  determined, 
the  protective  power  of  antitoxin  determined,  and  subsequently 
with  a  standard  antitoxin  any  new  toxin  may  be  titrated  after  the 
same  general  principles  as  described  previously  for  antitoxin  titra- 
tion.  By  this  method  the  presence  of  toxin  in  human  blood  may  be 
determined,  inasmuch  as  normal  human  serum  does  not  produce 
necrosis  upon  intracutaneous  injection  in  the  guinea-pig. 
Harriehausen  and  Wirth  found  that  the  serum  from  patients  suffer- 
ing with  diphtheria  produced  necrosis  owing  to  the  presence  of 
toxin,  and  this  was  demonstrated  in  five  cases  for  as  long  as  thirty- 
five  days  after  the  onset  of  the  disease.  By  the  use  of  a  titrated 
toxin  the  method  may  also  be  employed  for  determining  the  pres- 
ence and  amount  of  antitoxin  in  human  blood. 

Active  Immunization  Against  Diphtheria. — For  many  years  im- 
munization to  this  disease  was  entirely  in  the  form  of  passive  im- 
munization, practised  by  giving  protective  doses  of  antitoxin.  The 
antitoxin  was  given  in  doses  of  500  to  1000  units  and  served  to  pro- 
tect for  a  period  of  about  three  to  six  weeks.  This  was  of  special 
importance  in  institutions  and  families  exposed  to  the  disease.  The 
disadvantages  are  the  short  period  of  immunity  and  the  fact  that 
the  patient  may  thereby  become  hypersensitive  to  any  subsequent 
injections  of  horse  serum.  Active  immunity  had  been  observed  by 
Park  in  guinea-pigs  which  had  been  used  for  the  titration  of  anti- 
toxin, and  Park,  in  1905,  reported  that  horses  treated  with  neutral- 
ized mixtures  of  toxin  and  antitoxin  had  produced  immune  sera  as 
strong  as  400  units  per  c.c.  Theobald  Smith  suggested  a  similar 
method  of  immunization  in  man,  and  in  1913  von  Behring  reported 
the  successful  immunization  of  children  and  adults.  The  method 
of  immunization  with  toxin  and  antitoxin  mixtures  has  now  attained 
a  widespread  use  and  is  employed  even  as  early  as  the  fourth  day  of 
life.  Active  immunity  of  this  sort  is  demonstrable  by  the  Schick 
test  in  about  ten  days  after  treatment,  and  increases  so  that  in  the 
eighth  week  about  80  per  cent,  of  the  treated  individuals  are  immune, 
by  the  twelfth  week  96  per  cent,  are  immune,  and  at  the  end  of 
four  months  98  per  cent,  are  immune.  According  to  Park,  the  re- 
maining 2  per  cent,  become  immune  if  reinjected.  The  method  of 
immunization  is  to  give  three  injections  subcutaneously  one 
week  apart. 

For  the  preparation  of  the  mixture  a  ripe  toxin  is  used  and  so  diluted 
that  i.  c.c.  will  contain  200  minimum  lethal  doses  as  tested  against  guinea- 
pigs.  This  is  slightly  over-neutralized  with  antitoxin  and  the  mixture  should 
cause  no  symptoms  in  guinea-pigs  even  when  given  in  very  large  doses.  As 
indicated  above,  antitoxin  may  deteriorate  in  the  moist  state,  and  this  must 
beQavoided  in  the  toxin-antitoxin  mixtures.  If  the  mixtures  are  kept  at  about 
21  C.  the  mixture  remains  good  for  at  least  one  year,  although  it  is  prefer- 
able to  keep  it  at  a  lower  temperature.  The  injection  is  given  subcutaneously 
in  the  arm  at  the  insertion  of  the  deltoid  muscle.  The  immunity  developed 
following  injection  of  this  sort  is  against  toxin,  but  vaccination  against  diph- 


56  THE  PRINCIPLES  OF  IMMUNOLOGY 

theria  bacilli  themselves  may  also  be  practised  at  the  same  time  by  adding 
1000  millions  killed  organisms.  The  value  of  this  latter  procedure  has  not 
been  demonstrated  as  yet  and  it  is  not  widely  practised. 

The  advantage  of  the  use  of  toxin-antitoxin  mixtures  is  that  a  last- 
ing active  immunity  is  established.  It  has  been  suggested,  however, 
that  the  use  of  the  antitoxin  in  the  mixture  may  lead  to  the  de- 
velopment of  anaphylaxis.  It  has  been  maintained,  on  the  other 
hand,  that  the  mixture  of  the  toxin  probably  avoids  the  sensitizing 
effect  of  the  horse  serum,  but  this  is  not  borne  out  experimentally 
or  in  human  medicine.  Nevertheless,  the  amount  of  horse  serum 
given  is  relatively  small  and  the  development  of  hypersusceptibility 
in  man  following  these  small  amounts  is  not  very  common.  Park 
has  found  that  even  with  the  therapeutic  dose  of  diphtheria  antitoxin 
the  danger  of  anaphylaxis  is  extremely  small,  and  states  that 
among  330,000  cases  on  record  there  were  only  five  deaths.  As  we 
will  point  out  subsequently  (see  page  230),  the  reports  of  deaths 
following  antitoxin  administration  would  indicate  that,  in  a  certain 
percentage  at  least,  factors  other  than  anaphylaxis  are  operative. 
If  sensitiveness  to  horse  serum  is  known  it  is  suggested  that  anti- 
toxin prepared  in  some  other  animal,  such  as  the  goat,  may  be  em- 
ployed, but  sera  of  this  sort  are  not  easily  obtainable  in  the  market. 

Tetanus  Toxin  and  Antitoxin. — In  the  foregoing  consideration 
much  stress  has  been  laid  on  diphtheria  toxin  and  antitoxin,  with 
the  idea  that  the  problem  might  thus  be  presented  as  simply  as  pos- 
sible. Tetanus  toxin  and  antitoxin  have  been  studied  almost  if  not 
quite  as  intensively  as  diphtheria  and  deserve,  both  practically  and 
theoretically,  more  than  passing  mention.  The  important  facts  may 
be  given  briefly.  The  toxic  broth  produced  by  growth  of  bacillus 
tetani  contains  a  body  which  is  actively  hemolytic,  tetanolysin,  and 
more  important,  a  body  which  produces  the  symptoms  of  tetanus, 
tetanospasmin.  These  are  capable  of  specific  absorption,  so  that 
one  or  the  other  remains  free.  In  other  words,  the  tetanolysin  may 
be  removed  from  the  mixture  by  saturation  with  red  blood-cor- 
puscles, leaving  in  the  broth  the  tetanospasmin  which  may  be  in- 
jected with  the  production  of  symptoms  and  death.  An  antitoxin 
may  be  produced  specifically  for  the  tetanospasmin,  but  in  practice 
the  antitoxin  is  made  without  separation  of  the  two  toxins. 

The  toxin  is  produced  in  anaerobic  broth  cultures.  It  is  readily 
injured  by  heat  and  light,  and  is  best  preserved  in  the  dried  state. 
The  white  mouse  is  extremely  susceptible,  the  guinea-pig  less  so 
and  the  horse  somewhat  less,  whilst  pigeons  and  fowl  are  highly 
resistant.  The  antitoxin  is  produced  for  commercial  purposes  in  the 
horse,  the  earlier  doses  being  with  an  attenuated  toxin  following  a 
previous  injection  of  antitoxin.  The  serum  is  standardized  by  the 
use  of  white  mice  or  by  guinea-pigs,  the  procedure  being  practically 
the  same  as  for  standardization  of  diphtheria  antitoxin.  In  the 
United  States  the  toxin  is  used  as  a  standard.  It  is  precipitated  by 
ammonium  sulphate  and  dried.  The  minimum  lethal  dose  is  that 


TOXINS  AND  ANTITOXINS 


57 


which  kills  a  pig  of  350  grams  in  four  to  five  days.  The  antitoxic 
unit  is  ten  times  the  amount  of  antitoxin  necessary  to  protect 
against  100  minimum  lethal  doses. 

In  man  the  symptoms  appear,  as  a  rule,  first  in  trismus  of  the  jaw 
muscles,  but  in  experimental  animals  the  first  spasms  are  near  the 
injection  site  when  the  toxin  has  been  given  subcutaneously  or 
intramuscularly.  For  demonstration  purposes  the  dried  toxin  is 
freshly  dissolved  and  five  minimum  lethal  doses  injected  into  the 
thigh  muscles  of  one  hind  leg  of  a  guinea-pig.  In  the  course  of 
about  two  days  the  leg  is  found  stiff  and  extended,  the  animal  show- 
ing excitable  reflexes.  In  the  course  of  another  day  a  sudden  noise 
or  other  stimulus  will  excite  convulsions,  and  later  the  animal  will 
be  found  in  tonic  spasm  and  dies  with  all  four  extremities  in  exten- 
sion. If  the  toxin  is  given  intravenously  or  intraperitoneally,  the 
first  symptoms  are  excitable  reflexes,  then  general  clonic  and  finally 
tonic  spasm.  If  given  intracerebrally,  the  onset  is  by  epileptiform 
convulsions.  Rabbits  are  much  more  resistant  to  the  toxin,  and 
an  intravenous  injection  will  lead  to  gradual  wasting  and  a  cachectic 
death,  which  has  been  called  "  tetanus  sine  tetano."  The  suscep- 
tibility of  animals  varies  with  the  temperature  of  the  body.  Cold- 
blooded and  hibernating  animals  are  resistant  at  cold  winter 
temperatures,  but  become  susceptible  at  summer  temperatures. 

Tetanolysin.  The  tetanolysin  is  easily  demonstrable  in  a  toxin.  The  best 
red  blood-cells  for  use  are  those  of  the  goat,  sheep  and  horse.  The  fol- 
lowing protocol  will  show  the  method  of  titrating  the  tetanolysin.  The  toxin 
is  dissolved  so  as  to  make  a  I  per  cent,  solution  in  saline,  and  is  further  diluted 
for  the  experiment  1-2,  1-5,  i-io,  1-20.  The  blood-cells  are  washed  three 
times  and  suspended  in  salt  solution  so  as  to  make  a  5  per  cent,  solution.  For 
method  of  washing  red  blood-cells  see  page  118. 

Hemolysis 

Complete 

Complete 

Partial 

Slight 

None 

The  mixtures  are  incubated  in  a  water  bath  at  37°  for  one  hour. 

The  minimum  lytic  dose  in  the  above  instance  is  I  c.c.  of  a  1-5  dilution  of 
the  i  per  cent,  toxin  solution.  This  is  used  as  the  unit  to  determine  the 
antitetanolysin  in  an  antitetanic  horse  serum  as  in  the  following  protocol: 


Tube 

Toxin 

S  %  sheep  cells 

I 

1-2 

(i 

o 

c. 

c.) 

.0 

c.c. 

2 

i-5 

(i 

0 

c. 

c.) 

.0 

c.c. 

3 

I-IO 

(i 

0 

c. 

c.) 

.0 

c.c. 

4 

i  -20 

(i 

0 

c. 

c.) 

.0 

c.c. 

5 

Saline 

(i 

.0 

c 

c.) 

.0 

c.c. 

Tube                 Toxin 

Immune  serum           5  %  sheep  cells* 

Hemolysis 

I 

.0    C.C. 

-5 

dil. 

I-I,OOO      (l.O   C.C.) 

.0  c.c. 

None. 

2 

.0  c.c. 

-5 

dil. 

1-2,000     (i.o  c.c.) 

.0  c.c. 

None 

3 

.0    C.C. 

-5 

dil. 

I-IO,OOO    (l.O   C.C.) 

.0  c.c. 

Slight 

4 

.0    C.C. 

-5 

dil. 

1-20,000  (i.o  c.c.) 

.0  c.c. 

Partial 

5 

.0    C.C. 

-5 

dil. 

1-50,000  (i.o  c.c.)        ] 

.0  c.c. 

Complete 

Normal  horse  serum 

6 

.0    C.C. 

-5 

dil. 

I-IOO           (l.O    C.C.) 

.0  c.c. 

Complete 

7 

.0    C.C. 

-5 

dil. 

1-1,000      (l.O   C.C.) 

.0  c.c. 

Complete 

8 

.0  c.c. 

-5 

dil. 

None 

.0   C.C. 

Complete 

9        None 

I-IOO           (l.O    C.C.) 

.0   C.C. 

None 

*Add  the  sheep  cells  after  the  mixture  of  toxin  and  serum  has  been  incubated  for  one-half 
hour  and  then  incubate  one  hour.  For  method  of  diluting  serum  so  as  to  obtain  required 
strengths  see  page  84. 


58  THE  PRINCIPLES  OF  IMMUNOLOGY 

Tubes  6-9  are  controls  to  show  that  normal  horse  serum  is  not  antilytic, 
that  the  laking  dose  still  operates  after  the  preliminary  half-hour  incubation, 
and  that  horse  serum  itself  has  no  lytic  effect. 

Tetanospasmin.  For  the  demonstration  of  the  neutralization  of  tetano- 
spasmin  by  antitoxin  and  by  brain  substance,  the  following  experiments  are 
of  value.  Five  guinea-pigs  of  about  250  grams  are  needed. 

Pig  No.  i.    Inject  five  minimum  lethal  doses  of  toxin  into  the  thigh  muscles. 

Pig  No.  2.  Mix  ten  minimum  lethal  doses  of  toxin  with  one  unit  of 
antitoxin.  Allow  to  stand  at  room  temperature  for  about  twenty  minutes 
and  inject  as  in  pig  No.  I. 

Pig  No.  3.  In  a  sterile  mortar  grind  one-half  the  fresh  cerebrum  of  a 
guinea-pig  with  five  minimum  lethal  doses  of  toxin,  adding  salt  solution  in 
the  smallest  amount  necessary.  Allow  to  stand  two  hours,  centrifuge  and 
inject  the  supernatant  fluid  as  in  pig  No.  I. 

Pig  No.  4.  The  other  half  of  a  guinea-pig  brain  is  boiled  for  twenty 
minutes  in  water,  then  ground  up  with  five  minimum  lethal  doses  of  toxin, 
allowed  to  stand  two  hours,  centrifuged,  and  the  supernatant  fluid  injected 
as  in  pig  No.  i. 

Pig  No.  5  serves  as  a  control. 

The  guinea-pig  injected  with  toxin  will  show  typical  symptoms  as  de- 
scribed above,  beginning  with  'extension  of  the  leg  injected,  then  showing  excita- 
ble reflexes  followed  by  convulsions,  tetanic  spasm  and  death.  The  antitoxin 
and  fresh  brain  substance  will  protect  the  animals,  but  the  boiled  brain  will 
not.  The  normal  animal  serves  best  as  a  control  for  the  elicitation  of 
excitable  reflexes  and  slight  convulsions. 

Route  of  Absorption  of  Toxin. — It  is  of  interest  to  note  that  in 
man,  horse,  and  guinea-pig  the  central  nervous  system  alone  has 
the  power  of  neutralizing  tetanus  toxin,  but  in  the  case  of  the 
rabbit,  liver  and  spleen  in  addition  have  this  power.  It  is  main- 
tained that  the  gray  substance  of  the  nervous  system  possesses 
this  special  affinity,  and  the  white  matter  does  not.  Most  authorities 
believe  that  the  toxin  is  carried  along  nerve  tracks,  but  Zupnik 
maintains  that  it  travels  through  the  blood  stream  and  is  found  not 
only  in  the  nervous  system,  but  also  in  the  muscles.  Studies  of 
Meyer  and  Ransom  and  of  Marie  and  Teale  indicate  that  both 
routes  are  followed.  Depending  on  the  size  of  the  dose,  the  site  of 
inoculation,  and  perhaps  certain  other  factors,  one  or  the  other 
route  may  be  followed  predominantly,  but  never  to  the  exclusion  of 
the  other.  According  to  Teale  and  Embleton,  the  mode  of  transit 
along  nerve  trunks  is  by  way  of  the  axis  cylinders  and  the  peri- 
neural  lymphatic  vessels.  These  authors,  however,  maintain  that 
toxin  cannot  pass  from  the  choroidal  plexis  into  the  cerebrospinal 
fluid,  nor  from  the  capillaries  of  the  central  nervous  system  to  the 
nerve  tissues.  The  special  affinity  of  tetanospasmin  for  nerve  sub- 
stance is  not  peculiar  and  is  also  exhibited  by  the  neurotoxins  of 
snake  venom  and  by  the  toxin  of  bacillus  botulinus.  Teale  and 
Embleton  believe  that  tetanus  antitoxin  does  not  enter  the  sub- 
stance of  the  central  nervous  system  following  either  intravenous  or 
intrathecal  injection,  but  simply  acts  by  neutralization  of  the  toxin 
at  the  site  of  formation.  Clinical  experience  is  not  entirely  in 
agreement  with  the  experimental  work  of  these  authors,  since  cases 
have  been  improved  by  the  use  of  serum  after  tetanic  spasms 
have  appeared. 

Therapeutic  Use  of  Tetanus  Antitoxin. — As  is  well  known,  tetanus 


TOXINS  AND  ANTITOXINS  59 

follows  the  introduction  of  the  bacilli  or  their  spores  into  wounds  in 
such  a  fashion  that  anaerobic  growth  is  permitted.  The  incubation 
period  is  usually  considered  to  be  eight  days,  but  there  are  many 
variations  from  this  standard  period,  including  cases  that  have  an 
incubation  period  of  over  sixty  days.  The  mortality  from  the  dis- 
ease is  extremely  high,  the  average  ranging  between  78  and  90  per 
cent.  Its  incidence  in  civil  life  is  not  very  great,  but  in  time  of  war 
it  is  likely  to  occur  with  considerable  frequency  because  of  the  con- 
tamination of  war  wounds  by  soil  containing  the  organism  or  its 
spores.  In  the  American  Civil  War  the  disease  occurred  in  2.5  per 
cent,  of  the  wounded;  in  the  Franco-Prussian  War  in  3.5  per  cent.; 
and  in  the  World  War  6.5  per  cent.  In  the  earlier  wars  the  mortality 
ranged  between  80  and  90  per  cent.,  but  in  the  World  War,  owing 
in  all  probability  to  prophylaxis  and  treatment,  the  mortality  was 
50  per  cent.  In  carefully  studied  statistics  it  is  found  that  the  longer 
the  incubation  period  the  lower  is  the  rate  of  mortality.  This  general 
statement  held  true  before  the  use  of  anti-tetanic  serum  was  instituted 
and  still  holds  true.  The  difference  between  the  mortality  rate  of 
80  to  90  per  cent,  in  the  earlier  wars  and  50  per  cent,  in  the  World 
War  gives  an  excellent  illustration  of  the  decrease  in  mortality  that 
has  followed  the  introduction  of  serum  prophylaxis  and  treatment 
as  well  as  rational  surgery.  Knowing  that  the  organism  is  anaerobic 
in  growth,  surgery  demands  that  contaminated  wounds  be  kept  open 
for  the  access  of  air. 

Prophylactic  Use  of  Serum. — The  use  of  tetanus  antitoxin  is 
directed  toward  prophylaxis  and  toward  cure.  As  can  readily  be 
understood  from  the  experiments  outlined  above,  the  toxin  of  this 
disease  is  very  firmly  bound  to  nerve  tissues;  therefore,  treatment 
established  after  the  disease  has  appeared  is  not  likely  to  be  so 
effective  as  in  the  use  of  some  other  antitoxins.  Nevertheless,  not- 
able success  has  been  attained  in  some  cases  where  the  disease  has 
become  well  advanced  before  serum  treatment  has  been  instituted. 
Prophylactic  treatment  with  serum  is  given  as  early  after  the 
wound  as  possible,  and  in  both  military  and  civil  life  all  wounds 
contaminated  with  soil  should  receive  protective  doses  of  tetanus 
antitoxin.  This  is  given  subcutaneously  in  doses  of  500  to  1000 
units.  Wolff  reported  that  in  the  German  army  prior  to  December, 
1914,  prophylactic  injections  were  not  regularly  given  and  the  inci- 
dence of  tetanus  amounted  to  1.4  per  cent,  of  the  wounded.  During 
the  following  seven  months  prophylactic  injections  were  given  in 
the  field  to  all  those  wounded  by  grenades  and  shrapnel,  but  not 
those  wounded  by  rifle  bullets,  and  the  incidence  of  the  disease  was 
reduced  to  0.16  per  cent.  Protection  was  equally  as  successful  in 
the  Allied  armies,  and  instructions  were  given  to  administer  serum 
as  soon  after  injury  as  possible,  either  in  the  first-aid  station  or  in 
the  field  hospitals.  Experiences  in  the  British  army  demonstrated 
that  cases  might  develop  a  considerable  time  after  the  wound  was 
inflicted,  and  for  this  reason  subsequent  orders  directed  the  use  of 


6o  THE  PRINCIPLES  OF  IMMUNOLOGY 

500  units  of  tetanus  antitoxin  every  ten  days  for  four  doses.  Ic  was 
further  recommended  that  the  serum  be  given  subcutaneously  not 
more  than  seven  days  and  not  less  than  two  days  before  any  oper- 
ative procedure  upon  an  old  wound.  If  haste  is  necessary  the  serum 
may  be  given  intramuscularly  twelve  hours  before  operation.  Ob- 
viously this  suggests  the  possibility  that  organisms  may  remain 
dormant  in  wounds,  to  become  active  at  a  later  period ;  it  is  further  be- 
lieved that  the  antitoxin  is  probably  eliminated  in  about  ten  days, 
and  the  later  doses  of  immune  serum  are  given  in  order  to  neutral- 
ize any  toxin  that  might  be  produced  subsequently. 

Golla  tabulated  the  following  cases  not  receiving  prophylactic 
doses  of  antitoxin : 

Incubation  period  Cases  Mortality 

i-  7  days  17  (32.7  per  cent.)  82.5  per  cent. 

8-14  days  24  (46.2  per  cent.)  79.0  per  cent. 

15-21  days  6  (11.5  per  cent.)  54.0  per  cent. 

Over  21  days  5  (  9.6  per  cent.)  

This  shows  that  the  commonest  incubation  period  is  eight  to 
fourteen  days,  and  also  illustrates  the  fact  that  the  shorter  the  incu- 
bation period  the  more  serious  is  the  disease.  In  another  series  of 
patients  who  had  received  prophylactic  treatment  with  serum,  the 
following  data  were  collected ; 

Incubation  period  Cases  Mortality 

i-  7  days       6l  (22.6  per  cent.)     75.5  per  cent 
8-14  days       93  (34.6  per  cent.)     70.0  per  cent 


15-21  days  33     12.2  per  cent. 

21-30  days  19    7.05  per  cent. 

30-40  days  14       5.2  per  cent. 

40-50  days  9      3.3  per  cent. 

50-60  days  18      6.7  per  cent 

Over  60  days  22  (  8.2  per  cent.) 


/  w.w   J-/V-.1  WMt 

60.8  per  cent. 

62.8  per  cent. 

57.0  per  cent. 

33.4  per  cent. 

27.7  per  cent. 

40.8  per  cent. 


In  the  Franco-Prussian  War  only  5.7  per  cent,  exhibited  an  in- 
cubation period  of  more  than  twenty-one  days,  whereas,  according 
to  Golla,  in  the  last  war  30.54  per  cent,  showed  an  incubation  period 
of  more  than  twenty-one  days.  In  summary  it  may  be  stated  that 
the  introduction  of  prophylactic  injections  of  the  tetanus  antitoxin 
not  only  reduces  the  incidence  of  the  disease,  but  also  lengthens 
the  incubation  period,  and  therefore  reduces  the  mortality.  The 
delay  in  incubation  usually  leads  to  more  moderate  symptoms  as 
well  as  reduces  mortality,  and  oftentimes  the  cases  exhibit  tetanic 
spasms  in  only  one  extremity. 

Treatment  of  Tetanus  with  Serum. — When  the  disease  has  de- 
veloped, treatment  must  be  prosecuted  vigorously.  In  the  earlier 
years  of  its  employment  subcutaneous,  intramuscular,  and  intra- 
venous administration  was  practised,  but  arguing  from  the  nature  of 
the  disease  it  was  soon  suggested  that  intrathecal  injections  be  given. 
This  suggestion  was  followed  by  experimental  and  clinical  investi- 
gation, and  in  the  hands  of  the  majority  of  workers  the  method  has 
been  found  to  have  great  value.  Park  and  Nicoll  injected  twice  the 


TOXINS  AND  ANTITOXINS  61 

fatal  dose  of  toxin  into  the  hind  legs  of  guinea-pigs  and  seventeen 
to  twenty-four  hours  subsequently  injected  antitoxin  by  various 
routes.  Six  animals  receiving  the  immune  serum  subcutaneously 
died ;  fifteen  received  it  intracardially  and  two  survived,  whereas 
sixteen  animals  received  it  intrathecally  and  thirteen  survived.  It 
was  found  that  the  dose  necessary  for  intrathecal  injection  was  con- 
siderably smaller  than  the  dose  necessary  for  injection  into  the  cir- 
culation. Sherrington  conducted  a  similar  series  of  experiments 
upon  monkeys  with  essentially  the  same  results.  He  used  twenty- 
five  monkeys  for  his  series  of  injections  and  the  following  table 
gives  the  results ; 

Time  between 

Route  of  injection  giving  of  toxin     Recoveries     Deaths 

and  antitoxin 

Lumbar  intrathecal   47-78  hours  14  n 

Bulbar  intrathecal    47-78  hours  13  12 

Intravenous    47-7$  hours  7  18 

Intramuscular    47~78  hours  3  22 

Subcutaneous    47-78  hours  2  23 

Cerebral  subdural,  ten  cases  47~78  hours  o  10 

Clinically  Irons  was  not  able  to  demonstrate  such  a  marked  dif- 
ference in  results,  and  Leishman  and  Smallman  came  to  the  conclu- 
sion that  the  intramuscular  route  is  the  best.  The  work  of  Andrew 
and  Golla  demonstrated  the  clinical  value  of  the  intrathecal  method. 
Experimental  work  also  shows  that  although  antitoxin  can  take  up 
toxin  after  fixation  with  nerve  tissue,  such  a  release  of  toxin  is  re- 
stricted by  long  contact  with  the  nerve  tissue.  This  explains  the 
necessity  for  early  administration  of  antitoxin.  For  example,  the 
experiments  of  Doenitz  show  that  the  amount  of  antitoxin  neces- 
sary for  protection  increases  tremendously  with  a  lapse  of  time. 
He  injected  twelve  times  the  fatal  doses  of  toxin  and  found  that 
after  the  lapse  of  four  minutes  a  slight  excess  of  antitoxin  was  suffi- 
cient to  protect  the  animal,  but  after  eight  minutes  six  times  this 
dose  of  antitoxin  was  required ;  after  sixteen  minutes  twelve  times 
the  dose,  after  1  hour  twenty-four  times  the  dose;  in  four  to  six 
hours  six  hundred  times  the  original  dose,  and  after  six  hours  he 
was  unable  to  save  the  animals.  As  a  result  of  long  experience  with 
treatment  it  has  finally  been  determined  that  a  combination  of 
modes  of  injection  is  desirable  in  order  to  procure  complete  and 
lasting  saturation  of  the  body  with  antitoxin.  When  giving  intra- 
thecal injections  it  is  well  to  draw  off  the  spinal  fluid  and  then 
immediately  inject  3000  to  5000  units  of  toxin,  diluting  the  serum 
to  a  volume  of  10  to  15  c.c.  with  sterile  salt  solution.  Where  no 
fluid  can  be  withdrawn  from  the  spinal  canal  the  antitoxin  is  intro- 
duced very  slowly  by  gravity.  The  intrathecal  injection  is  further 
supplemented  by  10,000  to  15,000  units  intravenously,  and  three  to 
four  days  later  a  similar  injection  subcutaneously.  It  is  often  ad- 
visable to  repeat  the  intrathecal  injections  each  day  for  three  or 
four  days.  The  following  outline  taken  from  the  Memorandum 


62  THE  PRINCIPLES  OF  IMMUNOLOGY 

on  Tetanus  published  by  the  British  War  Office  gives  a  plan  for 
combined  injections  in  a  case  of  acute  tetanus; 

Day                     Subcutaneous  Intramuscular                  Intrathecal 

First 8,000  units  16,000  units 

Second   8,000  units  16,000  units 

Third    4,000  units               8,000  units 

Fourth 4,000  units              8,000  units 

Fifth    2,000  units  

Seventh    2,000  units  

Ninth    2,000  units  

This  outline  is  offered  as  a  suggestion  for  treatment  and  has 
been  applied  successfully.  The  doses  are  arranged  in  multiples  of 
8000  because  that  was  the  size  phial  issued  in  the  British  army. 
Doses  may  be  varied,  but  it  is  strongly  advised  to  administer  a 
total  of  75,000  to  100,000  units.  In  those  cases  with  long  incuba- 
tion period  the  dose  may  be  smaller,  and  if  the  case  is  one  of  spasm 
in  one  extremity,  without  evidence  of  involvement  of  higher  centers, 
such  as  spasm  of  jaw  muscles  (trismus),  the  serum  may  be  given 
by  intramuscular  and  subcutaneous  routes  in  amounts  of  3000  to 
6000  units.  The  patient  should  be  placed  so  that  he  lies  with  the 
feet  considerably  higher  than  the  head,  so  as  to  allow  drainage  to- 
ward the  head.  It  has  also  been  suggested  that  the  antitoxin  be 
applied  near  or  in  the  wound.  Calmette  recommended  that  pow- 
dered antitoxic  serum  be  applied  locally.  Suter  recommended  rub- 
bing the  fluid  serum  into  the  wound.  Bockenheimer  recommended 
that  it  be  introduced  in  the  form  of  ointment,  and  Robertson  satur- 
ated pads  of  cotton  with  antitoxin,  dried  these  for  twenty-four  hours 
at  40°  to  45°  C,  and  applied  them  locally.  As  will  be  seen,  these 
latter  measures  are  probably  more  in  the  nature  of  prophylaxis  than 
treatment,  and  no  definite  information  has  accrued  as  to  their  value. 
The  disadvantages  of  serum  treatment  are  essentially  the  same  as 
those  in  the  use  of  diphtheria  antitoxin,  but  in  addition  we  have  to 
deal  with  the  factor  of  introduction  of  foreign  serum  into  the  spinal 
canal.  This  frequently  leads  to  the  development  of  a  sterile  menin- 
gitis with  a  formation  of  purulent  fluid.  As  far  as  can  be  learned, 
this  inflammation  does  no  damage.  A  few  reports  of  nerve  and 
cord  lesions  following  the  use  of  antitetanus  serum  intrathecally 
have  been  reported,  but  they  are  extremely  small  in  number  com- 
pared to  the  number  of  cases  treated,  and  it  would  appear  that  the 
high  percentage  of  mortality  in  this  disease  justifies  the  intrathecal 
treatment  in  spite  of  the  minor  element  of  danger. 

Dysentery  Toxin  and  Antitoxin. — Dysentery  toxin  may  be  pro- 
duced in  broth  by  the  growth  of  the  Shiga  bacillus.  It  is  probable 
that  the  Flexner  and  Hiss-Russell  types  produce  only  an  endotoxin. 
This  is  consistent  with  the  greater  clinical  and  pathological  severity 
of  the  Shiga  type  of  dysentery.  The  broth  must  be  definitely  alka- 
line, the  optimum  stated  by  Doerr  being  reached  where  0.3  per  cent. 
soda  is  added  to  a  broth  neutral  to  litmus.  Rabbits  are  very  sus- 
ceptible and  the  intravenous  injection  of  a  filtered  toxin  broth  in 


TOXINS  AND  ANTITOXINS  63 

proper  doses  will  produce  marked,  often  bloody,  diarrhea,  wasting, 
paresis,  or  even  paralysis  of  extremities,  and  death.  The  autopsy 
shows  marked  inflammation,  often  hemorrhagic,  particularly  severe 
in  the  cecum,  but  also  involving  the  large  intestine  and  lower 
ileum.  Monkeys,  cats,  and  dogs  are  also  susceptible,  but  fowl,  pigeons, 
and  guinea-pigs  are  resistant.  Antitoxin  can  be  produced  in  horses 
and  goats.  There  is  considerable  difficulty  in  standardizing  such  a 
serum,  owing  to  the  variation  in  individual  susceptibility  of  ani- 
mals. Kraus  and  Doerr  have  shown  that  the  immune  serum  first 
shows  a  capacity  to  neutralize  toxin  in  vitro,  then  in  vivo  (simul- 
taneous injection  of  toxin  and  antitoxin  into  opposite  ear  veins), 
and  finally  it  attains  a  definite  curative  value  as  demonstrated  by 
primary  injections  of  toxin  followed  after  certain  time  intervals  by 
antitoxin.  A  serum  must  have  a  high  curative  value  before  it  is 
acceptable  and  is  used  in  doses  of  cubic  centimeters  rather 
than  units. 

Therapeutic  Use  of  Anti-dysentery  Sera. — After  the  discovery  of 
the  dysentery  bacillus  by  Shiga  in  1898  it  was  found  that  the  sepa- 
rate types  of  this  organism  vary  greatly  in  their  power  to  produce 
toxic  substances.  The  most  toxic  varieties  are  those  of  Shiga  and 
Kruse,  and  their  toxins  are  not  only  endotoxic  but  also  exotoxic  in 
nature,  a  fact  clearly  established  by  the  work  of  Todd,  Liidke, 
Kraus,  Doerr,  and  Rosenthal.  Shiga  was  the  first  to  immunize 
horses  with  killed  cultures  of  his  organism  and  produced  highly 
protective  sera  capable  of  saving  guinea-pigs  injected  with  six 
times  the  lethal  dose  of  living  bacilli.  This  specific  anti-bacterial 
serum  was  used  by  Shiga  with  encouraging  results  during  a  dysen- 
tery epidemic  in  Japan,  the  mortality  among  cases  treated  with 
Shiga's  serum  being  one-third  of  that  among  cases  treated  by  the 
usual  routine  procedures.  Not  only  was  the  mortality  greatly  re- 
duced, but  the  total  period  of  illness  decreased  from  forty  to  twenty- 
five  days.  A  similar  serum  was  prepared  by  Kruse  and  its  use 
reduced  the  mortality  among  Kruse's  cases  from  n  to  5  per  cent. 
Kraus  and  Doerr  also  obtained  favorable  results  from  the  use  of 
their  serum,  which  was  mainly  an  antitoxic  serum  produced  by  the 
injection  of  filtrates  of  young  cultures  into  horses.  Vaillard  and 
Dopter  treated  a  large  number  of  cases  with  a  serum  prepared  by 
themselves  and  possessing  both  antibacterial  and  antitoxic  prop- 
erties and  reported  highly  encouraging  results  with  a  mortality  of 
2  per  cent.,  while  the  mortality  otherwise  would  have  been  between 
ii  and  25  per  cent.  More  prompt  effects  were  obtained  when  the 
serum  was  given  at  the  earliest  moment  in  the  course  of  the  disease. 
Vaillard  and  Dopter  used  20  to  30  c.c.  in  moderate  cases  and  from 
40  to  80  c.c.  in  grave  cases.  In  late  cases  serum  injections  were 
often  of  value.  Graham  more  recently  has  added  a  valuable  contri- 
bution to  the  serum  therapy  in  bacillary  dysentery,  his  studies  being 
made  during  the  campaign  in  Macedonia.  Graham  used  a  serum 
prepared  at  the  Lister  Institute  and  gave  intravenous  injections  of 


64  THE  PRINCIPLES  OF  IMMUNOLOGY 

60  to  80  c.c.  once  or  twice  daily  during  the  first  three  days  of  treat- 
ment. Three  injections  were  followed  by  150  to  300  c.c.  of  saline 
daily  for  the  first  two  days  and  once  for  the  next  two  days,  the 
saline  injections  being  made  to  prevent  dehydration  of  the  tissues. 
In  mild  cases  no  saline  injections  were  necessary.  Most  of  the  cases 
arrived  after  the  third  day,  so  that  they  were  not  placed  under 
treatment  at  the  earliest  possible  moment.  On  entering  the  hos- 
pital all  cases  received  20  c.c.  of  the  serum  subcutaneously.  Klein 
also  maintains  that  anti-dysenteric  serum  given  early  and  in  large 
doses  intravenously  (60  to  100  c.c.)  is  efficacious.  He  found  that  the 
use  of  the  serum  produced  the  best  results  when  given  during  the 
first  five  or  six  days.  When  the  disease  has  entered  into  the  inter- 
mediate stage,  from  the  sixth  to  the  tenth  day,  the  outcome  of  the 
disease  is  irrespective  of  serum  treatment.  In  the  third  stage — 
tenth  day — the  use  of  serum  is  practically  without  value.  Waller 
treated  140  cases  with  the  Lister  Institute  serum  and  found  that 
the  early  use  of  the  serum  resulted  in  shortening  the  duration  of  the 
disease.  He  gave  three  subcutaneous  injections  of  140  c.c.  at  eight- 
hour  intervals  during  the  first  twenty-four  hours  to  fairly  severe 
cases.  Rosenthal,  in  a  series  of  serum-treated  cases,  found  a  mor- 
tality of  0.6  per  cent.  In  other  units  the  mortality  was  10  to  15  per 
cent.  Sixty  c.c.  of  sera  were  given  by  Rosenthal  on  the  first  day, 
followed  by  40  to  60  c.c.  on  the  second,  and  if  no  improvement  was 
observed  subsequent  doses  of  40  c.c.  were  given  up  to  a 
total  of  400  c.c.  Usually  the  stools  were  free  of  blood  in  forty-eight 
hours,  and  their  number  reduced  from  60  to  15  or  10  per  day. 
Lantin  also  thinks  that  the  use  of  serum  constitutes  an  efficient 
specific  method  of  treatment.  He  gave  the  serum  by  rectum  in  doses 
from  30  to  50  c.c.  Neumann  used  human  convalescent  serum  in  400 
cases.  Intestinal  irrigations  with  silver  solutions  were  also  em- 
ployed by  this  author.  Only  six  of  his  cases  ended  fatally.  Jacob, 
on  the  other  hand,  and  with  him  also  Nolf,  failed  to  obtain  success 
with  serum  therapy  in  this  disease.  Jacob  treated  ninety  cases, 
using  polyvalent  sera  and  injecting  subcutaneously  or  intravenously 
doses  ranging  from  20  to  400  c.c.  during  the  first  or  second  week  of 
the  disease.  According  to  the  British  Medical  Research  Com- 
mittee, serum  treatment  of  bacillary  dysentery  is  not  satisfactory. 
Nevertheless,  numerous  investigators  showed  that  this  method  of 
treatment  has  a  well-established  clinical  value  as  expressed  in  the 
view  of  Schittenhelm,  who  states  that  it  should  be  employed  in  all 
cases  of  more  than  three  or  four  days*  duration,  and  in  all  cases 
showing  toxemia  and  severe  symptoms,  and  in  cases  where  the 
number  of  stools  are  more  than  twelve  in  the  course  of  twenty-four 
hours.  It  should  be  given  early  in  the  disease  and  in  massive  doses. 
If  possible  the  type  of  the  infecting  organism  should  be  known  prior 
to  the  administration  of  these  massive  doses.  This  can  readily  be 
done  in  twenty-four  hours  in  a  well-equipped  laboratory.  The 
serum  used  should  be  polyvalent,  because  there  are  a  number  of 


TOXINS  AND  ANTITOXINS  65 

serologically  distinct  types  of  dysentery  bacilli.  Schmitz,  for  in- 
stance, found  in  a  dysentery  outbreak  among  prisoners  of  war  in 
Roumania  strains  which  resembled  the  Shiga  bacillus  but  were 
serologically  entirely  distinct  types.  Pribram  also  found  that  an 
antitoxin  specific  for  the  Shiga-Kruse  toxin  is  inactive  toward  the 
toxin  of  a  strain  D118H  (Hallmann).  Furthermore,  the  curative 
action  of  anti-dysentery  serum  is  due  first  to  its  content  in  antitoxin, 
and  second  to  its  anti-bacterial  properties. 

The  serum  can  further  be  employed  for  prophylactic  injections  in 
doses  from  10  to  30  c.c.,  but  the  immunity  thus  produced  will  be  only 
of  a  short  duration.  Recently  Boehneke  and  Elkeles  have  inocu- 
lated over  100,000  persons  with  a  polyvalent  bacillary  toxin-anti- 
toxin preparation  called  dys-bakta,  but  complete  protection  was  not 
secured.  It  was  noted,  however,  that  infections  occurring  in  the 
inoculated  individuals  were  usually  of  slight  severity,  and  death  a 
very  unusual  occurrence.  The  reaction  following  inoculations  was 
no  more  severe  than  that  following  typhoid  inoculations.  Immunity 
thus  produced  lasted  for  at  least  three  months. 

Botulinus  Toxin  and  Antitoxin. — Bacillus  botulinus  produces  a 
toxic  body  leading  to  symptoms  often  called  "  ptomaine  poisoning." 
The  toxin,  however,  is  apparently  independent  of  the  medium  used, 
is  destroyed  by  moist  heat  of  58°  C.  for  three  hours,  and  of  80°  C. 
for  one-half  hour,  and  is  capable  of  inducing  the  formation  of  an 
antitoxin.  The  symptoms  produced  by  the  toxin  are  marked  in- 
crease or  decrease  of  saliva  flow,  vomiting,  sometimes  diarrhea, 
but  more  often  constipation,  often  retention  of  urine,  paralysis  of 
eye  muscles,  aphoria,  rarely  fever  or  disturbance  of  sensitivity. 
Death  frequently  ensues  following  the  appearance  of  symptoms  of 
bulbar  paralysis  with  disturbances  of  respiration  and  heart  action. 
The  necropsy  shows  marked  general  passive  congestion  and  throm- 
bosis of  the  meningeal  vessels  sometimes  with  slight  hemorrhage. 
Unlike  other  toxins,  that  of  botulism  resists  the  digestive  juices  and 
is  absorbed  by  way  of  the  alimentary  canal.  It  can  be  neutralized 
by  brain  substance  and  by  the  lipoids,  lecithin,  cholesterol,  and  by 
fats,  such  as  butter  and  oil.  It  is  toxic  for  man,  monkey,  cat,  rabbit, 
and  guinea-pig. 

The  Use  of  Immune  Sera  in  Botulism. — Van  Ermengem  in  1895 
discovered  the  cause  of  botulism  poisoning  to  be  an  exotoxin  pro- 
duced by  a  strictly  anaerobic  Gram-positive  bacillus  which  he  iso- 
lated from  portions  of  a  ham  that  had  caused  fifty  cases  of  poisoning 
at  Ellezelles,  Belgium.  The  disease  has  an  exceptionally  high  mor- 
tality of  almost  loo  per  cent.,  and  up  to  the  present  time  the  per- 
centage of  fatal  cases  has  been  as  great  as  it  was  fifty  years 
ago.  The  reason  for  this  lies  in  the  fact  that  the  early  symptoms  of 
the  disease  are  not  recognized  until  the  toxemia  is  well  established. 
In  the  year  1897  Kempner  showed  that  susceptible  animals  may  be 
successfully  immunized  to  the  toxins  of  this  organism  and  obtained 
a  potent  antitoxin  from  goats,  I  c.c.  of  the  serum  protecting  against 
5 


66  THE  PRINCIPLES  OF  IMMUNOLOGY 

100,000  minimal  lethal  doses.  Forssman  and  Lundstrom  were  also 
successful  in  their  immunization  attempts,  using  attenuated  toxins. 
Wassermann  immunized  horses  and  produced  sera  of  undeniable 
value  in  animal  experiments.  In  this  country  sera  were  prepared 
by  Graham,  Brueckner  and  Pontius,  Buckley,  Hart,  Meyer,  Hurwitz 
and  Taussig,  Burke,  Dickson,  and  Howitt  mainly  for  experimental 
purposes,  using  rabbits,  sheep,  goat,  cattle,  and  dogs  for  immuniza- 
tion. According  to  Dickson  and  Howitt,  laboratory  experiments 
show  that  the  antitoxin  may  protect  against  the  action  of  the  toxin 
for  at  least  twenty-four  hours  after  the  administration  of  one  test 
dose  of  toxin,  but  the  effectiveness  is,  to  a  certain  extent  at  least, 
dependent  upon  the  amount  of  toxin  injected.  Like  tetanus  anti- 
toxin, botulinus  antitoxin  should  be  given  early  if  it  is  to  be  effec- 
tive, and  even  in  well-established  cases  it  is  strongly  advisable  to 
give  antitoxin  in  massive  doses,  because  Kob  has  demonstrated  that 
this  toxin  may  persist  in  the  blood  nine  days  after  the  poisoning. 
If  symptoms  of  botulism,  such  as  hypersecretion  of  mucus  from 
mouth  and  nose,  visual  disturbances,  aphonia,  dysphagia,  and 
paralysis  of  the  intestinal  tract  appear,  antitoxin  should  be  admin- 
istered as  soon  as  possible,  and  should  be  given  in  large  doses 
intravenously.  Dickson  also  advises  the  use  of  antitoxin  to  all 
persons  who  have  eaten  fowl  that  have  suffered  from  limberneck. 
Of  importance  is  the  use  of  polyvalent  sera  because  of  the  discovery 
of  Leuchs  that  two  strains,  the  one  of  Van  Ermengem  and  a 
Darmstadt  strain,  were  distinct,  that  the  toxin  of  one  was  not 
affected  by  the  specific  antitoxin  of  the  other,  and  vice  versa.  As 
for  the  effect  of  botulinus  antitoxin  in  man,  little  is  known,  as  it 
has  been  used  only  in  isolated  instances.  Dickson  and  Howitt,  in 
1918,  gave  85  c.c.  of  immune  goat  serum  (i  c.c.  equivalent  to  3000 
M.L.D.  for  a  guinea-pig)  to  each  of  two  patients  at  Madera,  Cali- 
fornia. Both  patients  recovered,  but  as  the  antitoxin  was  given  very 
late,  in  fact,  after  all  the  more  seriously  poisoned  patients  had 
succumbed,  there  is  no  definite  evidence  that  the  course  of  the  ill- 
ness was  favorably  influenced  by  the  antitoxin,  although  it  was 
later  shown  that  the  toxin  of  the  strain  recovered  from  the  food  was 
Type  A.  McCaskey  used  small  doses  of  antitoxin  in  three  patients 
(5  to  10  c.c.).  One  died  and  two  recovered  and  this  author  there- 
fore thinks  the  serum  to  be  of  some  aid.  Nonnenbruch  obtained 
rapid  improvement  in  his  case  after  the  use  of  antitoxin.  His  pa- 
tient became  poisoned  after  eating  sausage.  Jennings,  Haas  and 
Jennings  in  the  recent  Detroit  outbreak  used  Graham's  serum  in  a 
dose  of  42  c.c.  intravenously  in  one  case  without  apparent  effect,  and 
20  c.c.  in  two  injections  to  another  patient,  who  recovered,  and  state 
that  the  latter  case  was  not  of  mild  type.  Dickson  and  Howitt 
found  that  of  all  the  outbreaks  in  which  the  serum  had  been  used, 
with  the  exception  of  the  cases  of  McCaskey,  the  toxin  was  that  of 
Type  A,  and  consequently  when  Type  B  serum  was  used  it  could 
not  be  expected  to  give  any  satisfactory  results.  As  it  is  impos- 


TOXINS  AND  ANTITOXINS  67 

sible  to  determine  quickly  the  type  of  toxin  in  a  particular  outbreak, 
it  is  of  the  greatest  importance  to  use  polyvalent  sera. 

Gas  Bacillus  Toxin. — The  frequent  occurrence  of  gas  gangrene 
in  the  Great  War  has  given  especial  interest  to  the  preparation  of 
antitoxins  for  the  organisms  causing  the  disease.  Klose,  in  1916, 
and  Bull  and  Pritchett,  in  1917,  were  able  to  prepare  a  soluble  toxin 
of  the  bacillus  Welchii  or  as  it  is  often  named  bacillus  perfringens. 
Bull  and  Pritchett  drew  especial  attention  to  the  necessity  for  select- 
ing a  strain  which  is  capable  of  producing  toxin  in  fairly  large 
amounts.  The  British  Medical  Research  Committee  reports  that 
the  toxin  of  vibrion  septique  has  very  little  effect  following 
subcutaneous  injection.  Upon  intravenous  injection,  however,  it 
produces  convulsions  and  usually  death  in  a  few  minutes.  An 
antitoxin  may  be  produced,  but  it  is  not  effective  after  the  toxin  has 
been  injected.  The  toxin  of  bacillus  edematiens  produces  massive 
edema  about  the  site  of  inoculation.  The  toxin  of  bacillus  aerogenes 
capsulatus  was  found  to  have  a  necrotic  action  upon  the  tissues ;  it  is 
generally  toxic  in  large  doses  and  animals  may  be  protected  by 
antitoxic  serum. 

The  Use  of  Immune  Sera  in  Gas  Gangrene — Treatment  of  the 
Disease. — Leclainche  and  Vallee,  Sacquepee,  Weinberg  and  Seguin, 
Bull  and  Pritchett  were  the  first  to  apply  serum  therapy  in  wounds 
infected  with  the  gas  bacilli.  Leclainche  and  Vallee's  and  Weinberg 
and  Seguin's  serums  were  polyvalent  and  also  antibacterial,  while 
Bull  and  Pritchett's  serum  was  antitoxic.  In  1917  Bull  and 
Pritchett  produced  an  exotoxin  from  twenty-four-hour  cultures  of 
bacillus  aerogenes  capsulatus,  which  when  injected  into  pigeons 
or  guinea-pigs  caused  local  edema,  necrosis,  and  hemolysis  of  red 
cells,  and  was  capable  of  stimulating  the  formation  of  an  antitoxin. 
Bull's  claim  for  the  potency  of  his  antitoxic  serum  was  based  on  ex- 
periments in  which  he  used  pure  cultures  of  bacillus  aerogenes  cap- 
sulatus and  made  no  attempt  to  discriminate  between  the  different 
types  of  the  organism,  such  as  have  been  found  to  exist  by  Henry, 
or  to  consider  the  fact  that  in  war  wounds  the  bacillus  aerogenes 
capsulatus  is  not  the  only  causal  factor  of  gas  gangrene.  From 
Nevin's  work  it  would  appear  that  neither  anti-perfringens  serum 
(bacillus  aerogenes  capsulatus  anti-microbial  serum)  nor  Bull's  anti- 
toxin afford  any  protection  when  other  pathogenic  anaerobes  inci- 
dent to  war  wounds  are  present,  together  with  bacillus  aerogenes 
capsulatus,  whereas  when  the  vibrion  septique  and  bacillus 
edematiens  are  present  in  mixed  infections  without  bacillus  aero- 
genes capsulatus,  the  prophylactic  use  of  the  specific  sera,  even 
when  diluted  by  another  serum,  is  effective.  Weinberg  and  Seguin, 
who  have  contributed  extensively  to  the  serum  therapy  of  gas 
gangrene,  found  treatment  by  serum  alone  limited  because  of  rapid 
absorption  of  toxin  in  this  disease.  The  association  of  rational 
surgery  and  of  serum  therapy  gives  the  best  results.  In  a  series  of 
sixty-six  cases  reported  by  these  authors  in  which  sixty  did  not 


68  THE  PRINCIPLES  OF  IMMUNOLOGY 

receive  serum  treatment,  three  received  non-specific  treatment  and 
three  suffered  complications,  thirty-five  deaths  were  recorded,  while 
in  a  series  of  twenty-four  specifically  treated  cases  only  five  deaths 
occurred,  thus  reducing  the  mortality  from  more  than  55  per  cent, 
to  less  than  21  per  cent.  The  serum  used  in  these  cases  was  poly- 
valent, produced  against  bacillus  aerogenes  capsulatus,  the  vibrion 
septique,  and  bacillus  edematiens.  Duval  and  Vaucher,  in  1917, 
reported  fifty  cases  in  which  a  combination  anti-perfringens,  anti- 
edematiens,  and  anti-vibrion  septique  serum  prepared  by  Weinberg 
and  Seguin  was  injected  prophylactically.  In  none  of  these  patients 
did  gas  gangrene  develop,  although  all  were  of  the  most  severely 
wounded  type.  Twenty-five  died  as  a  result  of  severe  multiple 
wounds  without  any  signs  or  symptoms  of  gas  gangrene. 

Prophylactic  Use  of  Sera. — A  year  later  these  same  authors  re- 
ported a  series  of  281  cases  in  which  severely  wounded  patients 
were  injected  with  polyvalent  serum  prepared  at  the  Pasteur  Insti- 
tute. Eighteen  developed  gas  gangrene  (6.4  per  cent.),  and  of  these 
ten  died,  resulting  in  a  mortality  of  3.5  per  cent.,  the  usual  mortality 
from  gas  gangrene  in  severely  wounded  being  16  per  cent.  Mairesse 
and  Regnier  found  among  1016  wounded  men  examined  bacteriologi- 
cally  297  gas  bacillus  infections.  They  received  prophylactic  injec- 
tions of  anti-serum  depending  on  the  type  of  organism  present.  In 
thirty  instances,  or  10  per  cent,  of  the  cases,  the  disease  developed. 
Ivens  also  used  Weinberg  and  Seguin's  serum  in  222  cases  for 
prophylactic  injections.  Among  these  no  deaths  occurred,  and 
fourteen  amputations  were  performed  without  fatal  results.  With 
Leclainche  and  Vallee's  serum  (154  cases)  four  died,  and  in  fifty- 
seven  other  cases  treated  with  both  sera  two  deaths  occurred. 
Further  favorable  reports  were  made  by  Quenu,  Bazy  and  Routier, 
Vincent  and  Stodel,  Marquis,  Dufour  and  Samelaigne.  Curative 
injections  were  given  by  Duval  and  Vaucher  with  20.7  mortality. 
Rouvillois,  Guillaume,  Louis,  Pedeprade,  and  Thibierge  treated 
twenty-five  cases,  five  of  whom  died.  Of  these  three  were  moribund 
on  entrance  to  the  hospital.  Mairesse  and  Regnier's  thirty  treated 
cases  had  a  mortality  of  16.6  per  cent. 

Van  Beuren,  who  reports  a  personal  communication  from  Lieut.- 
Col.  W.  Elser,  states  that  prophylactic  doses  were  given  to  15,000 
soldiers  and  controlled  by  15,000  others.  According  to  this  finding 
there  was  not  sufficient  difference  in  the  incidence  rate  to  warrant 
any  definite  declaration  as  to  the  protective  value  of  the  sera  used. 
Apparently  these  investigators  were  favorably  impressed;  for  they 
laid  the  failure  to  secure  better  results  to  the  weakness  of  the 
serum  then  available.  Elser  advises  the  following  routine  for  the 
serum  treatment : 

1.  A   prophylactic   dose   of   polyvalent   serum,    combined   with 
tetanus  antitoxin,  given  as  early  as  possible  after  the  receipt  of 
the  wound. 

2.  Bacteriologic  examination  of  the  wound  and  establishment  of 


TOXINS  AND  ANTITOXINS  69 

the  presence  of  gas  bacillus  infection   and  determination   of  the 
variety  of  the  bacteria. 

3.  Administration  of  specific  serum,  either  single  or  polyvalent 
or  "  pooled,"  according  as  there  are  one  or  more  gas  formers  found, 
and  also  the  administration  of  anti-streptococcus  serum,  since  the 
latter  organism  is  very  commonly  found  in  association  with  the 
other  organisms. 

From  the  general  reports  obtained  during  the  Great  War  it  is 
considered  that  intravenous  injection  is  to  be  preferred,  in  combina- 
tion with  deep  muscular  injections  in  the  vicinity  of  the  wound. 
From  these  reports  it  seems,  then,  that  the  use  of  a  polyvalent  anti- 
bacterial and  antitoxic  serum  is  advisable,  but  much  work  on  the 
subject  must  yet  be  done.  From  all  the  observations  at  hand  it  is 
safe  to  state  that  the  best  results  are  to  be  obtained  from 
preventive  injections. 

Bacterial  Hemotoxins. — As  an  example  of  the  hemotoxins  pro- 
duced by  bacteria  certain  details  of  staphylolysin  may  be  consid- 
ered. The  hemotoxin  is  produced  by  twelve  to  thirteen  days' 
growth  of  staphylococcus  pyogenes  aureus  or  albus  in  broth.  The 
organisms  are  killed  and  the  broth  filtered  through  a  porcelain 
filter.  The  filtrate  can  be  preserved  by  the  addition  of  5  per  cent,  of 
a  solution  made  up  of  10  parts  phenol,  20  parts  glycerol,  and  70  parts 
water.  Doses  of  0.025  to  0.05  c.c.  should  completely  hemolyze  one 
drop  of  rabbit  blood  after  two  hours  at  37°  C.  Antilysin  may  be 
produced  by  immunization  of  animals  and  is  found  normally  to  a 
slight  extent  in  normal  human  blood  and  in  that  of  certain  lower 
animals.  The  victims  of  staphylococcus  infections  frequently  show 
an  increased  antilysin  content  of  the  serum.  This  fact  has  been  em- 
ployed by  Bruck,  Michaelis,  and  Schultze  to  diagnose  staphylococcus 
infections,  some  cases  showing  increases  of  ten  to  one  hundred  times 
over  the  normal  antilysin.  The  simplicity  of  bacteriological  exami- 
nation, however,  makes  this  method  of  diagnosis  by  comparison 
rather  cumbersome  and  time  consuming.  Whether  or  not  antilytic 
sera  would  be  of  value  in  the  treatment  of  those  cases  that  resist  or 
are  unsuitable  for  vaccine  treatment  has  not  been  determined  so  far 
as  we  have  been  able  to  learn. 

PHYTOTOXINS 

Introduction. — Although  literally  the  phytotoxins  include  all  the 
toxins  of  vegetable  origin  the  term  usually  is  restricted  to  include 
those  originating  in  forms  of  vegetable  life  higher  than  the  bacteria. 
With  this  definition  thought  would  be  first  directed  to  the  poison- 
ous fungi,  but  as  has  already  been  shown,  only  one  of  the  poisons  so 
far  isolated  is  capable  of  inducing  antibody  formation.  The  poison- 
ous elements  of  poison  ivy  and  poison  oak  produce  no  antibodies. 
The  poisonous  elements  of  those  plants  that  produce  "  hay  fever  " 
require  separate  discussion,  because  the  toxic  factor  operates  only 
on  individuals  who  show  a  peculiar  susceptibility  or  "  hypersus- 


70  THE  PRINCIPLES  OF  IMMUNOLOGY 

ceptibility."  The  element  of  hypersusceptibility  in  this  connection 
will  be  deferred  until  after  the  presentation  of  the  fundamental 
material  on  anaphylaxis  and  hypersusceptibility.  The  following 
paragraphs  will  present  briefly  the  essentials  concerning  ricin,  abrin, 
robin,  crotin,  curcin,  and  phasin.  This  brevity  is  justified  by  the  rela- 
tively small  practical  importance  of  these  substances. 

Ricin  is  the  toxic  principle  of  the  castor-oil  bean,  ricinus  com- 
munis.  It  was  isolated  by  Gibson  in  1887  and  named  ricin  by 
Stillmark  in  1888.  Gushing  made  very  strong  toxic  preparations  and 
Field  states  that  ricin  will  kill  rabbits  in  doses  of  o.oooi  mg.  per 
kilo ;  guinea-pigs,  0.0008  mg. ;  dogs,  0.0006  mg. ;  cats,  0.0002  mg. ;  and 
goats,  0.003  mg.  Following  injection  there  is  an  incubation  period 
succeeded  by  diarrhea,  somnolence,  weakness  of  extremities,  and 
death.  At  the  necropsy  are  found  reddening  and  swelling  of  Peyer's 
patches,  mesenteric  and  retinal  hemorrhages,  ulcers  of  stomach, 
nephritis,  general  lymphatic  swelling,  and  softening  and  degenera- 
tion of  the  pyramidal  cells  of  the  cerebral  cortex.  Beauvisage  re- 
ported 150  cases  of  ricin  poisoning  in  man  of  which  nine  were  fatal. 
Many  of  these  were  children  who  ate  the  seeds,  but  there  were  also 
soap  makers  who  handled  the  beans  in  soap  manufactories.  Ricin 
and  the  other  toxins  in  the  group  may  be  precipitated  with  the  pro- 
teins by  ammonium  sulphate ;  they  are  precipitated  by  alcohol  and  are 
gradually  destroyed  by  proteolytic  enzymes.  Jacoby,  however, 
claims  to  have  produced  ricin  and  abrin  which  failed  to  give  pro- 
tein reactions.  Osborne,  Mendel,  and  Harris  maintain  that  ricin  is 
inseparably  associated  with  protein,  and  that  Jacoby's  error  was 
due  in  all  probability  to  the  fact  that  he  obtained  a  product  so  toxic 
that  the  small  amounts  necessary  for  toxic  action  were  too  small  to 
give  the  protein  reactions.  The  most  striking  character  of  ricin  in 
vitro  is  its  capacity  to  agglutinate  the  red  blood-corpuscles  of  prac- 
tically all  warm-blooded  animals.  It  may  agglutinate  other  body 
cells,  precipitates  protein,  and  is  adsorbed  by  casein,  fibrin,  coagu- 
lated serum  albumin,  and  by  silk.  Jacoby  concludes  that  ricin  is  a 
mixture  of  agglutinin  and  toxin,  the  two  having  certain  molecular 
groups  in  common.  Ehrlich  believes  that  these  may  undergo  altera- 
tion into  agglutinoid  and  toxoid.  The  mechanism  of  the  agglutina- 
tion is  not  clear  and  many  hypotheses,  none  quite  satisfactory,  have 
been  advanced.  Ehrlich  produced  an  antiricin  by  giving  increasing 
doses  to  animals  by  mouth,  and  then  changing  to  subcutaneous  in- 
jections. This  antiricin  was  used  by  Ehrlich  in  the  development  of 
much  of  his  hypothesis  of  the  toxin  antitoxin  union  because  of  the 
ease  of  manipulation  as  compared  with  the  time-consuming  and 
expensive  method  of  working  with  animal  injections  of  toxin  anti- 
toxin mixtures.  In  addition  to  the  antitoxin  there  are  present  in  the 
serum  a  closely  related  antiagglutinin  (with  which  Ehrlich  worked) 
and  a  precipitin  for  ricin  solutions. 

Abrin  is  obtained  from  paternoster  or  jequirity  bean,  abrus  pre- 
catorius,  and  was  described  by  Warden  and  Waddell  in  1884.  It  is 


TOXINS  AND  ANTITOXINS  71 

much  less  toxic  than  ricin,  producing  gastro-enteritis,  hemorrhages, 
and  swelling  of  lymph-nodes.  Local  applications  led  to  an  acute 
conjunctivitis  and  in  hairy  regions  to  transitory  loss  of  hair,  both 
of  which  may  be  protected  against  by  immunization.  Robert  states 
that  in  India  and  Ceylon  cattle  were  immunized  (by  feeding  beans) 
against  the  effects  of  wounds  by  abrin-coated  projectiles.  Roemer 
found  that  by  repeated  application  to  the  conjunctival  sac  of  one 
eye,  he  could  produce  an  immunity  which  first  protected  that  eye 
and,  after  further  immunization,  served  to  protect  the  opposite 
untreated  eye,  in  this  later  stage  becoming  a  general  immunity  with 
antiabrin  in  the  serum.  Abrin  is  also  a  hemagglutinating  agent, 
and  can  be  distinguished  from  ricin  by  immunological  experiment. 
In  many  respects  abrin  and  its  immunity  resemble  ricin  very  closely. 

Crotin  is  derived  from  croton  seed,  croton  tiglium,  and  is  less 
toxic  than  either  ricin  or  abrin.  According  to  Elfstrand,  it  agglu- 
tinates the  red  blood-corpuscles  of  beef,  sheep,  swine,  and  frog;  it 
hemolyzes  the  cells  of  rabbit,  cat,  and  crow,  and  has  no  effect  on  the 
erythrocytes  of  man,  dog,  guinea-pig,  rat,  hen,  goose,  and  pigeon. 
Immune  sera  can  be  produced  by  the  usual  methods.  Jacoby  found 
in  Grubler's  pepsin  a  body  which  he  called  pseudo-anticrotin,  cap- 
able of  neutralizing  the  action  of  crotin  on  erythrocytes  in  vitro  but 
not  in  vivo,  and  he  found  the  same  substance  in  gastric  and 
intestinal  mucosa. 

Curcin  is  produced  from  the  seeds  of  jatropha  curcus,  and  robin 
from  the  leaves  and  bark  of  robinia  pseudacacia.  Immune  sera  can 
be  produced  against  both  of  these. 

Phasin  is  a  name  given  by  Landsteiner  and  Raubitschek  to  a 
hemagglutinating  substance  found  in  the  seeds  of  the  bean,  pea, 
lentil,  and  vetsch.  Antiagglutinins  are  found  in  normal  serum  and 
may  be  increased  experimentally,  but  this  substance  or  group  of 
substances  can  hardly  be  regarded  as  belonging  to  the  class  of 
toxins  because  of  little  or  no  toxic  symptoms  following  injection. 

Pollen  Proteins  or  Pollen  Toxin. — The  modern  studies  of  hay 
fever  and  of  asthma  place  this  subject  so  clearly  in  the  group  of 
anaphylactic  phenomena  that  its  consideration  is  deferred  (see 
page  233). 

ZOOTOXINS 

Introduction. — The  zootoxins  include  the  poisonous  elements 
produced  in  animal  life.  They  may  be,  and  most  frequently  are,  in 
the  form  of  excretions  of  special  poison  glands  or  are  found  in  secre- 
tions of  other  glands,  in  blood  and  in  tissues.  The  most  important 
are  the  snake  poisons,  but  there  are  also  included  the  poisons  of 
spiders,  scorpions,  bees,  centiped.es,  tarantula,  toads,  poisonous  fish, 
duck-bill  platypus,  and  the  sera  of  various  animals. 

The  snake  venoms  differ  somewhat  in  their  action  according  to 
family,  the  colubridae,  including  the  cobra,  Australian  black  snake, 
and  others ;  the  viperidae,  including  the  European  viper  and  Ameri- 


72  THE  PRINCIPLES  OF  IMMUNOLOGY 

can  rattlesnake ;  and  the  hydrophinse  or  poisonous  sea  snakes.  The 
venoms  secreted  by  special  glands  are  injected  during  the  bite 
through  fine  canals  in  the  fangs  (not  the  forked  tongue),  and  are 
all  hemolytic.  The  fact  that  the  blood  of  snakes  contains  poisons 
similar  to  those  of  the  venom  indicates  that  the  poison  glands 
secrete  with  little  alteration  the  poison  of  the  blood.  Never- 
theless, snake  bites  may  be  poisonous  for  snakes  of  other  species, 
and  also  for  other  members  of  the  same  species.  Geoffroi 
and  Hunauld,  in  1737,  and  Fontana,  in  1781,  noted  the  anticoagu- 
lant action  of  venom,  but  the  work  of  Weir  Mitchell  in  1860, 
and  of  Weir  Mitchell  and  E.  T.  Reichert  in  1886,  served  as  the  great- 
est stimulus  to  modern  investigation.  Mitchell  and  Reichert 
showed  that  the  venom  of  the  rattlesnake  produces  rapid  coagula- 
tion of  the  blood  and  death,  but  that  if  the  animal  survives  the  blood 
is  reduced  in  coagulability.  C.  J.  Martin  confirmed  this  in  regard  to 
Australian  snakes  and  showed  that  the  phenomenon  could  be  con- 
trolled by  dosage  of  the  venom.  In  addition  to  hemolysis  and 
alteration  of  coagulation,  other  properties  are  present,  and  Flexner 
and  Noguchi  showed  in  venom  the  presence  of  hemotoxins,  in- 
cluding hemolysins  and  hemagglutinins,  leucocytolysins,  and  an 
endotheliotoxin  which  they  named  hemorrhagin.  Pearce  showed 
that  hemorrhagin  produced  lysis  of  endothelium  leading  to  hemor- 
rhage. In  addition,  venoms  contain  proteolytic  enzymes,  invertase, 
lipase,  and  probably  certain  ferments  dealing  with  coagulation. 
Martin  found  fibrin  ferments  which  probably  aid  in  thrombus  for- 
mation. Lamb  found  that  even  citrated  blood  could  be  clotted  by 
venoms.  Negrete  found  the  anti-coagulating  element  closely  asso- 
ciated with  the  proteins  of  the  venom.  Morowitz  claims  the  pres- 
ence of  an  antithrombokinase.  Modern  studies  by  Houssay 
Sordelli  and  Negrete  with  the  venoms  of  fourteen  snakes,  Indian, 
American,  and  Australian,  show  that  clotting  time  does  not  parallel 
closely  the  dose  of  venom,  that  venoms  clot  whole  blood,  plasma, 
and  fibrinogen  solutions,  and  that  mammalian  blood  is  more  sus- 
ceptible than  that  of  birds,  batrachians,  and  snakes.  The  addition 
of  citrate,  oxalate,  magnesium  sulphate,  hirudin,  and  peptone  delay 
the  action  of  the  venom,  the  oxalate  acting  the  most  intensely.  It 
seems  likely  that  large  doses  of  venom  bring  to  bear  a  sufficient 
amount  of  fibrin  ferment  to  produce  clotting  and  that  the  later 
effects  are  due  to  the  anti-coagulating  power  of  the  venom  after  the 
fibrin  ferment  is  exhausted.  Inasmuch  as  the  hemolysis  of  venom 
is  somewhat  closely  related  in  mechanism  to  hemolysis  in  general, 
it  will  be  discussed  in  the  chapter  on  Hemolysis  (see  page  141). 
Venom  toxins  resemble  other  toxins  in  that  they  are  precipitated 
with  proteoses,  whilst  the  factor  which  produces  local  irritation 
comes  down  with  globulin,  although  Faust  maintains  that  the  active 
principles  of  venoms  are  glucosides.  Venom  toxins  are  destroyed 
by  heat,  the  cobra  poisons  as  a  class  by  100°  C.,  and  the  viper 
poisons  by  85°  C.  They  do  not  dialyze  and  deteriorate  under  the 


TOXINS  AND  ANTITOXINS 


73 


influence  of  light,  radium,  and  oxiding  agents.  There  is  an  incuba- 
tion period  and  the  venoms  are  definitely  and  specifically  antigenic. 

Venoms  act  in  extremely  small  amounts.  The  fatal  dose  of 
cobra  venom  for  man  is  probably  o.oi  to  0.03  gm.,  rattlesnake  venom 
0.15  to  0.3  gm.,  and  poisonous  sea  snakes  o.ooi  to  0.003  Sm->  or  ten 
times  as  toxic  as  cobra  venom.  The  bite  of  the  cobras  produces 
little  pain  and  local  reaction,  probably  due  to  its  small  content  (2 
per  cent.)  of  globulin,  which  contains  the  local  irritant  property  of 
the  venom.  A  feeling  of  stiffness  spreads  from  the  region  of  the 
wound,  followed  by  vertigo  and  weakness  of  muscles  of  locomotion, 
tongue,  jaw,  esophagus,  and  preservation  of  senses,  resembling  a 
very  acute  bulbar  palsy  with  death  in  a  few  hours.  Gushing,  how- 
ever, finds  that  the  action  of  the  poison  is  particularly  upon  motor 
nerve  termini.  The  venom  of  the  vipers  produces  a  marked  local  reac- 
tion, probably  due  to  its  large  (25  per  cent.)  globulin  content,  with 
pain,  swelling,  local  bleeding,  blood  in  the  serous  membranes  and 
hematuria.  Nausea  and  vomiting,  excited  reflexes,  and  even  con- 
vulsions are  followed  by  prostration,  paraplegia  of  lower  extremities 
which  extends  upward  and  resembles  an  acute  ascending  spinal 
paralysis  with  death  in  one  to  three  days.  Langmann  states  that, 
"  if  the  patient  recovers  from  the  paralysis,  a  septic  fever  may  de- 
velop ;  not  rarely  there  remain  suppurating  gangrenous  wounds 
which  heal  poorly."  The  suppuration  of  snake  bites  (viperidae) 
has  been  the  subject  of  considerable  study;  Welch  and  Ewing 
ascribed  this  to  loss  of  bactericidal  property  of  the  blood  after  venom 
poisoning.  Flexner  and  Noguchi  demonstrated  a  loss  of  the  com- 
plement of  the  blood,  an  element  necessary  to  its  full  bactericidal 
power.  They  believed  that  the  complement  was  used  up  by  the 
venom  whose  amboceptors  require  complement  for  their  action, 
therefore  leaving  little  or  none  free  for  the  bactericidal  amboceptors. 
Morgenroth  and  Kaya  claim  that  the  complement  is  actually  de- 
stroyed by  the  venom.  Of  considerable  importance  in  favoring  infec- 
tions must  be  the  local  necrosis  of  tissue  caused  by  the  venom  and  the 
associated  hemorrhage,  aided  by  the  customary  radical  surgery  of 
the  wound. 

The  production  of  antisera  was  placed  on  a  practical  basis  by 
Calmette  in  1894  and  Frazer  in  1895.  Calmette  attenuated  cobra 
venom  for  the  first  four  injections  by  the  addition  of  equal  volumes 
of  i  per  cent,  gold  chloride  solution,  and  then  gave  small  doses  of 
the  native  toxin,  gradually  increasing  until  a  powerful  antivenin 
was  developed.  Phisalix  and  Bertrand  attenuate  viper  venom  by 
heating  the  first  dose  to  75°  C.  and  then  after  two  days  giving  one- 
half  the  minimum  lethal  dose  of  toxin.  It  was  at  first  thought  that 
the  antivenin  produced  by  cobra  venom  would  protect  against  all 
venoms,  but  it  was  soon  shown  that  the  sera  were  specific  for  the 
venom  employed.  Antivenin  also  neutralizes  that  element  of  venom 
which  induces  loss  of  bactericidal  power  of  the  blood.  Noguchi  has 
shown  that  the  antivenin  of  rattlesnake  venom  neutralizes  the 


74  THE  PRINCIPLES  OF  IMMUNOLOGY 

hemorrhagin.  Such  sera  also  contain  precipitins  for  the  proteins  of 
the  special  venoms  employed  and  for  the  serum  proteins  of  the 
same  species  of  snake.  These  are  highly  but  not  absolutely  specific. 
The  mechanism  of  venom-antivenin  union  is  probably  very  closely 
similar  to  that  of  toxin-antitoxin  unions  of  other  varieties,  although 
Kyes  holds  that  the  former  is  distinctly  in  the  nature  of  the  chemi- 
cal reaction  between  a  strong  acid  and  a  strong  base. 

Scorpion  poison  is  secreted  by  special  glands  in  the  abdomen.  In 
human  adults  the  symptoms  are  rarely  severe,  except  for  marked 
local  reaction,  but  it  is  stated  that  the  bite  of  an  African  scorpion 
may  kill  children.  As  a  rule,  the  most  serious  effects  are  from  the 
subsequent  infection  of  the  wounds.  Todd  was  able  to  prepare  a 
specific  immune  serum  for  the  poison  of  scorpions.  According  to 
Houssay,  scorpion  venom  acts  pharmacologically  much  as  veratrin ; 
it  is  a  smooth  muscle  stimulant.  He  states  that  serum  therapy  is 
useful  and  specific. 

Spider  poison  is  secreted  by  glands  in  the  thorax.  The  common 
spiders  are  not  venomous,  except  the  "  cross  spider  "  whose  venom, 
much  weaker  in  the  saliva  than  in  the  ovaries,  closely  resembles 
snake  venoms  in  chemical  properties  and  agglutinin,  and  probably 
contains  a  neurotoxin.  Sachs  prepared  an  antivenin  against  this 
venom.  Some  of  the  larger  spiders  are  extremely  poisonous,  par- 
ticularly the  Malmigatte  of  southern  Russia  and  related  species  in 
South  America  and  Africa.  Large  numbers  of  cattle  have  been 
poisoned  with  as  high  as  12  per  cent,  mortality,  but  the  bite  is 
rarely  fatal  for  man. 

The  tarantula  produces  a  poison  which  operates  almost  entirely 
locally,  and  it  is  stated  that  an  antitoxin  can  be  produced  against  the 
Russian  tarantula. 

Centipedes. — Certain  centipedes  secrete  a  poison  in  special  glands 
that  discharge  through  the  claws,  capable  of  producing  considerable 
local  reaction.  But  one  case  of  fatal  poisoning  has  been  reported 
from  Texas,  that  of  a  child  four  years  old. 

Bees,  wasps,  and  hornets  secrete  a  poison  closely  similar  for  all 
three.  Bee  poison  contains  formic  acid  and  in  addition  a  poison 
which  does  not  give  the  usual  protein  reactions,  but  is  destroyed 
by  proteolytic  enzymes ;  it  resists  heat  to  100°  C.,  weak  acids,  and 
alkalies.  The  poison  contains  a  hemolysin  which  operates  in  much 
the  same  manner  as  does  cobra  hemolysin.  The  bite  produces 
marked  local  reaction,  but  only  in  cases  of  extreme  hypersuscep- 
tibility  are  there  general  effects  or  death.  Part  of  the  lack  of 
severity  of  bee  poison  is  due  to  the  small  dose  injected,  for  if  col- 
lected in  large  amounts  and  injected  intravenously  into  dogs  it  can 
produce  death.  The  resistance  of  professional  bee  keepers  to  the 
bites  is  probably  due  to  the  fact  that  repeated  bites  lead  to  de- 
velopment of  immunity,  although  it  is  possible  that  the  doctrine  of 
the  survival  of  the  fittest  may  play  its  part.  Ants  probably  pro- 


TOXINS  AND  ANTITOXINS  75 

duce  a  somewhat  similar  poison  in  addition  to  formic  acid.  The 
"  black  flies  "  of  the  woods  produce  a  poison  not  as  yet  identified, 
but  no  poison  has  as  yet  been  isolated  from  the  mosquito.  Numer- 
ous other  insects  appear  to  have  poisonous  secretions,  but  as  yet  no 
studies  have  been  made  in  detail  as  to  their  isolation  and  identification. 

Toads,  frogs,  and  salamanders  produce  dermal  secretions  which 
are  poisonous,  several  of  which  operate  like  digitalis  and  some  like 
epinephrin.  These  poisons  are  interesting  from  a  pharmacological 
point  of  view,  but  as  they  are  not  capable  of  producing  immune  re- 
actions in  animals  they  deserve  no  extensive  discussion  here. 

Poisonous  fish  comprise  several  groups.  One  group  secrete 
poisons  in  special  glands,  the  poison  being  discharged  through 
spines.  Such  poisons  contain  a  hemolysin  which  requires  an 
activator,  as  in  the  case  of  cobra- venom  hemolysins.  These  poisons 
act  as  powerful  local  irritants  and  as  cardiac  depressants  and  may 
cause  death.  Only  one  variety  of  fish  produces  poisoning  by  its 
bite,  the  poison  being  secreted  in  the  gums.  Other  fish  are  poison- 
ous when  eaten  even  when  quite  fresh,  the  poison  being  found 
especially  in  the  ova  and  ovaries.  The  symptoms  may  be  of  a 
severe  choleriform  type  frequently  fatal,  or  of  a  less  severe  gastro- 
intestinal type,  not  commonly  leading  to  death.  Certain  fish,  par- 
ticularly in  the  tropics,  rapidly  decompose  with  the  formation  of 
poisonous  products  or  ptomains.  The  bites  of  crabs  may  produce 
peculiar  erysipelas-like  lesions  or  "  erysipeloid,"  but  the  origin  and 
nature  of  the  poison  are  not  known.  Many  individuals  develop 
toxic  symptoms  after  eating  shell-fish  and  other  sea  food,  in  some 
cases  due  to  the  decomposition  of  the  food,  but  in  most  instances 
due  to  a  peculiar  hypersusceptibility  which  will  be  discussed  under 
Hypersusceptibility  (see  page  230). 

Eel  serum  deserves  special  consideration  because  of  the  fact  that 
immune  bodies  can  be  produced.  It  is  not  poisonous  when  ingested, 
but  is  highly  so  if  given  intravenously  and  it  produces  conjunctivitis 
when  instilled  into  the  sac.  Relatively  large  doses  lead  to  rapid 
death  and  small  doses  may  produce  cachexia  and  death  after  sev- 
eral days.  The  toxic  'element  is  in  the  albumin  fraction  of  the 
serum  and  is  destroyed  by  58°  C.  for  fifteen  minutes.  It  contains 
a  hemolysin  and  probably  also  a  neurotoxin.  The  hemolysin  does 
not  act  as  an  amboceptor,  reactivation  by  fresh  serum  being  im- 
possible after  the  eel  serum  has  been  heated.  Immune  sera  can  be 
produced  which  neutralize  the  hemolysin  in  vitro  and  also  protect 
animals  from  death  by  the  eel  serum.  The  serum  of  lampreys  and 
rays  is  similarly  toxic. 

The  parasitic  protozoa  and  other  animal  parasites  are  strikingly 
free  from  substances  which  induce  immunity.  The  protozoa  show 
few  exceptions  to  this  rule.  Cytolysins  can  be  produced  experi- 
mentally for  amebse,  but  no  such  reaction  takes  place  in  human 
patients.  Active  immunity  to  trypanosome  infection  can  be  pro- 


76  THE  PRINCIPLES  OF  IMMUNOLOGY 

duced,  and  it  is  claimed  that  immunity  can  be  conferred  passively. 
The  trypanosomes,  however,  can  become  immune  to  trypanocides. 
Malarial  parasites  produce  among  other  things  a  hemolysin,  but 
there  is  no  indisputable  evidence  that  immunity  occurs  in  malaria, 
nor  have  immune  reactions  been  developed.  Sarcosporidia  of  sheep 
produce  a  toxin  fatal  for  rabbits  in  doses  of  o.oooi  gm.,  against  which 
an  antitoxin  may  be  produced  in  rabbits.  Complement-fixation  re- 
action is  positive  in  infested  sheep.  Man  may  be  infested  by  the 
cyst  of  one  tape  worm,  the  tenia  echinococcus,  the  cyst  contents 
being  definitely  toxic,  as  shown  when  a  cyst  ruptures  into  a  body 
cavity,  e.g.,  the  peritoneum.  Serum  of  infested  patients  contains  a 
precipitin  for  the  cyst  proteins  and  also  a  complement-fixing  body, 
Zapelloni  reporting  93  per  cent,  positive  complement-fixations  in  500 
cases  examined.  Of  the  adult  tape  worms  which  infest  man  the 
dibothriocephalus  latus  is  the  most  important  from  the  immunologi- 
cal  standpoint,  although  this  parasite  is  rare  in  America.  The 
proglottids  contain  a  thermostabile  hemolytic  lipoid  liberated  on 
the  death  of  the  segments  by  auto-digestion.  There  is  also  a  ther- 
molabile  hemagglutinin.  It  is  probable  that  the  hemolysin  is 
either  associated  with  other  cytolysins  or  that  a  species  cytolysin  is 
present  which  also  acts  as  a  hemolysin.  This  is  responsible  for  the 
primary  type  of  anemia  seen  in  dibothriocephalus  latus  patients. 
The  serum  of  these  patients  contains  a  precipitin  for  the  fluid  ob- 
tained by  antolytic  digestion  of  the  segments.  Of  the  nematodes 
the  ascaris,  the  trichinella  spiralis,  the  hook  worm,  and  certain  forms 
of  filaria  have  been  investigated.  Certain  ascarids  produce  poison- 
ous substances  without  immunological  relations.  In  regard  to 
trichinosis  Salzer  has  found  that  the  serum  of  recovered  patients 
has  distinct  therapeutic  value  in  infested  patients  and  protects  ani- 
mals against  experimental  infestation.  Complement-fixation  has 
been  found  to  be  of  value  in  the  diagnosis  of  trichinosis.  It  has  been 
claimed  that  the  anemia  of  hook-worm  infestation  is  due  to  a 
hemolytic  poison,  but  there  is  doubt  that  this  is  as  important  as 
the  small  repeated  hemorrhages  produced  by  the  bite  of  these  para- 
sites. There  is  little  of  immunological  significance  in  the  studies  of 
the  filariae.  The  guinea-worm  (filaria  medinensis)  contains  in  its 
body  a  violent  irritant  which  may  be  discharged  by  rupture  of  the 
worm  during  forcible  attempts  at  its  removal,  and  leads  to  severe 
local  inflammation  and  even  to  gangrene. 

Mammalia  do  not  produce  poisons  except  in  the  somewhat  ques- 
tionable case  of  the  male  duck-bill  platypus  of  Australia,  a  survivor 
of  the  very  earliest  forms  of  mammalian  life.  Special  glands  are 
said  to  secrete  a  poison  like  that  of  Australian  snakes,  which  is  dis- 
charged through  a  hollow  movable  spur  on  the  hind  foot.  There  is 
serious  question  as  to  the  toxic  properties  of  this  secretion,  certain 
authorities  believing  that  the  sequences  of  such  wounds  are  due  to 
infection  and  that  the  secretion  is  of  importance  only  as  a  secondary 
sex  character.  The  serum  of  certain  mammals  is  toxic  on  injection, 


TOXINS  AND  ANTITOXINS  77 

as,  for  example,  beef  serum,  which  in  doses  of  0.5  c.c.  will  kill  a 
guinea-pig  in  a  few  minutes.  Dog  serum  is  also  toxic  for  guinea- 
pigs  in  somewhat  larger  doses  (i.o  to  2.0  c.c.).  Horse  serum  is  toxic 
for  cats  in  doses  of  i.o  c.c.  per  kilo,  and  for  guinea-pigs  in  doses  of 
20.0  c.c.  per  kilo.,  but  man  is  practically  insusceptible,  except  in 
those  cases  of  hypersusceptibility  in  which  small  doses  of  serum 
produce  serious  symptoms  and  even  death.  Such  toxic  sera  con- 
tain hemolysins  and  agglutinins  in  small  amounts  and  reduce  the 
coagulability  of  the  blood,  but  death  is  probably  due  to  other  factors. 
Except  for  cases  of  natural  or  artificial  hypersusceptibility,  the  toxic 
element  is  destroyed  by  heat  of  56°  C.  and  is  removed  by 
animal  charcoal. 


CHAPTER  V 
AGGLUTININS  AND  PRECIPITINS 

GENERAL  INTRODUCTION. 
AGGLUTININS. 

BACTERIAL  AGGLUTINATION. 
INTRODUCTION. 

PRODUCTION  OF  IMMUNE  AGGLUTININS. 
PRELIMINARY  TITRATION. 

BLEEDING  THE  IMMUNE  RABBIT. 

METHODS  OF  TITRATION. 
MACROSCOPIC. 

MICROSCOPIC  (THE  WIDAL  TEST). 
SPECIFICITY  OF  AGGLUTININS. 

GROUP  REACTIONS. 

ABSORPTION  OF  AGGLUTININS. 
INHIBITION  ZONES. 
INFLUENCE  OF  HEAT. 
INFLUENCE  OF  ELECTROLYTES. 
INFLUENCE  OF  HYDROGEN  ION  CONCENTRATION. 
THE  MECHANISM  OF  AGGLUTINATION. 

ALTERATIONS  OF  CELL  AGGLUTINABILITY. 

THE  NATURE  OF  AGGLUTININS. 

PHYSICAL  BASIS  OF  AGGLUTINATION. 

THE   DREYER   TEST. 
HEM  AGGLUTININS. 

HETERO-HEMAGGLUTININS. 
ISO-HEMAGGLUTININS. 

CLASSIFICATION    OF    HUMAN    ISO-HEMAGGLUTININS. 
CHARACTERS. 
MECHANISM. 

ISO-HEMAGGLUTININS   IN   OTHER  ANIMALS. 
BLOOD  TRANSFUSION. 

METHODS  FOR  TESTING  HUMAN  BLOOD. 
REACTIONS. 

CHEMICAL  AGGLUTINATION  OF  ERYTHROCYTES. 
CONGLUTI  NATION. 
PRECIPITINS. 

INTRODUCTION. 

NATURE  OF  REACTION. 

EXPERIMENTAL  DEMONSTRATION. 

DELICACY  OF  REACTION. 

INFLUENCE  OF  HEAT  AND  OTHER  AGENTS. 

PRACTICAL    APPLICATION. 

THE  FORENSIC  BLOOD  TEST. 
BIOLOGICAL  RELATIONSHIPS. 

ORGAN    SPECIFICITY. 
DETECTION   OF   FOOD  ADULTERATION. 
FUNCTION  IN  IMMUNITY. 

General  Introduction. — Jf  a  clear  albuminous  urine  be  boiled  the 
invisible  protein  aggregates  clump  together,  become  visible  as 
flocculi,  and  sink  to  the  bottom  of  the  test-tube.  If  to  a  colloidal 
suspension  of  mastic  be  added  a  proper  concentration  of  common  salt 
a  similar  flocculation  of  the  mastic  occurs.  Red  blood-corpuscles  or 
bacteria  shaken  in  physiologic  salt  solution  form  a  cloudy  suspen- 
sion of  particles  invisible  to  the  naked  eye.  They  may  be  clumped 
together  by  a  variety  of  methods  in  similar  flocculi  which  become 
78 


AGGLUTININS  AND  PRECIPITINS 


79 


clearly  visible  as  small  particles  and  sink  to  the  bottom  of  the  tube 
more  quickly  than  would  the  individual  cells  in  the  original  suspen- 
sion. The  first  example  is  one  of  precipitation  and  the  last  of 
agglutination.  In  the  immunological  sense,  precipitation  implies 
flocculation  of  a  protein  solution  by  means  of  specific  antibodies,  so 
that  large  aggregates  are  formed  and  thrown  out  of  solution.  Simi- 
larly the  term  agglutination  signifies  clumping  together  by  means 
of  specific  antisera  of  cells  originally  in  smooth  emulsion,  so  that 
the  clumps  are  visible  microscopically  or  grossly,  and  sink  rapidly 
to  the  bottom  of  the  containing  vessel.  Animals  may  be  immunized 
to  a  protein  in  solution,  as,  for  example,  blood  serum  or  egg  white, 
so  that  the  animal's  serum  contains  a  body,  the  precipitin,  capable 
of  precipitating  the  protein  used  for  immunization.  Similarly  bac- 
teria, red  blood-corpuscles,  or  even  other  cells  may  be  injected  re- 


FlG.  3. — Wooden  box  for  holding  rabbits  during  injections  into  or  bleeding  from  the  ear  vein. 

0 

peatedly  into  animals  leading  to  the  formation  within  the  animal  of 
a  body,  the  agglutinin,  appearing  in  the  blood  serum  and  capable  of 
clumping  the  type  of  cell  injected.  These  phenomena,  although 
closely  related  and  probably  fundamentally  identical  in  nature,  will, 
for  eminently  practical  reasons,  be  discussed  separately. 


AGGLUTININS 

Bacterial  Agglutination. — Although  others  had  observed  the 
phenomenon  of  agglutination,  Gruber  and  Durham,  in  1896,  were 
the  first  to  study  it  intensively  in  the  course  of  work  on  the  colon 
bacillus  and  the  cholera  vibrio.  They  pointed  out  the  specificity  of 
the  reaction  and  the  fact  that  it  differed  in  certain  essentials  from 
previously  studied  serum  reactions.  These  points  will  be  discussed 


So 


THE  PRINCIPLES  OF  IMMUNOLOGY 


in  some  detail,  but  it  must  be  pointed  out  at  once  that  the  specificity 
is  not  absolute.  It  was  soon  found  that  blood-cells  and  later 'other 
body  cells  could  be  agglutinated  by  specific  sera.  It  was  also 
found  that  agglutinins  of  various  kinds  exist  normally  in  certain 
sera,  these  being  called  normal  agglutinins  as  opposed  to  the  arti- 
ficially produced  or  immune  agglutinins.  It  was  found  that  the 
agglutinins  resist  heat  of  56°  C,  a  degree  sufficient  to  destroy  com- 
plement, and  that  after  being  rendered  inactive  by  heat  cannot  be 
reactivated  by  fresh  normal  serum.  It  was  soon  observed  that  in 
the  course  of  infectious  disease  due  to  a  specific  organism  agglu- 
tinins^are  likely  to  develop,  and  this  led  to  the  discovery  in  Widal's 


FIG.  4. — Method  of  obtaining  blood  from  the  posterior  auricular  vein  of  the  rabbit's  ear.     The 

vein  has  been  incised  by  means  of  a  small  hypodermic  needle.    The  same  position  of  the  animal 

serves  for  intravenous  injections  which  are  given  into  the  posterior  auricular  vein. 

clinic  in  Paris,  a  few  months  after  Gruber  and  Durham's  publica- 
tion, of  the  now  widely  used  Widal  reaction  for  typhoid  fever.  Con- 
versely with  a  serum  of  known  type,  the  antigenic  bacteria  may  be 
identified.  The  demonstration  of  agglutination  may  be  by  the 
microscopic  method  or  by  the  macroscopic  method.  In  our  pre- 
sentation of  the  subject  it  is  considered  desirable  to  illustrate  the 
points  by  actual  experiment,  and  for  this  reason  we  proceed  to  take 
up  the  method  of  producing  immune  agglutinins  in  the  laboratory 
and  subsequently  present  the  factors  which  qualify  and  modify 
the  process  of  agglutination. 

Production  of  Immune  Agglutinins. — Injections  for  producing 
agglutinins  may  be  subcutaneous   intraperitoneal,  intravenous,  or  a 


AGGLUTININS  AND  PRECIPITINS 


81 


combination  of  these,  using  first  the  subcutaneous  or  intraperitoneal 
routes  followed  later  by  intravenous  injections.  Bacteria  are  usu- 
ally killed  by  heat  or  chemicals  before  injection,  although  after  im- 
munization is  well  under  way  living  organisms  may  be  employed. 
The  use  of  living  organisms  is  often  of  service  in  the  development 
of  a  serum  of  high  titer. 


POUPACTd  U  a. 


PIG.  5. — Method  of  complete  bleeding  from  the  femoral  vessels  of  the  rabbit  (see 
text  page  83). 

The  following  will  serve  as  a  fairly  typical  example  of  the  process  of 
immunization  for  the  production  of  an  anti-typhoid  agglutinin.  The  cultures 
used  are  twenty-four  hour  agar  slants  inoculated  by  zig-zagging  the  loop 
back  and  forth  over  the  surface  so  as  to  have  the  surface  well  covered  by 
growth.  A  measured  amount,  5.0  c.c.  or  10.0  c.c.  of  sterile  salt  solution  is 
added,  the  tube  allowed  to  stand  ten  or  fifteen  minutes  and  then  vigorously 
rotated  between  the  palms  of  the  hands.  This  procedure  gives  a  much 
smoother  emulsion  than  washing  off  by  sucking  in  and  blowing  out  from  a 
pipette  or  by  scraping  off  with  a  platinum  loop  and  is  less  susceptible  to 
6 


82  THE  PRINCIPLES  OF  IMMUNOLOGY 

contamination.  The  suspension  is  pipetted  into  a  sterile  tube  and  the  growth 
killed  by  placing  in  a  water  bath  of  not  less  than  56°  C.  or  more  than  60°  C. 
for  two  hours.  Rabbits  are  desirable  animals  because  of  the  ease  of  intra- 
venous injection.  For  ease  in  handling,  the  animal  is  placed  in  a  box  as  shown 
in  Figs.  3  and  4.  The  ear  is  shaved  along  the  course  of  the  posterior  auricular 
vein  situated  near  the  posterior  margin  of  the  ear  on  its  upper  surface,  is  cleansed 
with  soap  and  water  and  sponged  over  with  alcohol.  Usually  the  alcohol  makes  the 
vein  stand  out  prominently,  but  if  it  does  not,  the  ear  may  be  pinched  near  its  root 
so  as  to  distend  the  vein,  or,  if  necessary,  brushed  over  lightly  with  a  sponge  dipped 
in  xylol.  Xylol  should  be  used  very  sparingly,  because  of  the  danger  of  an 
inflammation,  which  may  make  subsequent  injections  difficult.  Bleeding  from 
the  puncture  may  be  stopped  by  pinching  the  ear  for  a  few  moments  at  the 
site  of  injection.  Usually  one  ear  is  used  for  injections  and  the  other  for 


PIG.  6. — A  flask  placed  upright  after  blood  has  clotted 

with  oblique  surface.     The  serum  drains  to  the  bottom 

of  the  flask  and  is  easily  withdrawn. 

test  bleeding.  The  earlier  injections  or  bleedings  are  near  the  tip  of  the  ear, 
the  later  ones  approaching  the  base.  The  following  protocol  illustrates 
an  immunization: 

Day  Killed  typhoid  emulsion 

I  0.05  agar  slant  * 

6  o.i     agar  slant 

ii  0.2    agar  slant 

16  0.2     agar  slant 

21  0.2     agar  slant 

*  If  10  c.c.  saline  had  been  added  to 
the  culture,  0.5  c.c.  suspension  would 
contain  0.05  agar  slant. 

Preliminary  Titration. — One  week  after  the  last  injection  0.5-1.0  c.c.  blood 
is  withdrawn  from  an  ear  vein  and  the  serum  separated  and  titrated  for  the 
agglutinin.  (See  Fig.  4.)  If  the  titer  is  not  satisfactory,  the  immunization 
may  be  continued  with  living  bacilli  as  follows  : 

Day  Living  typhoid  emulsion 
I  0.05  agar  slant 

4  o.i     agar  slant 

8  0.2     agar  slant 


AGGLUTININS  AND  PRECIPITINS 


After  7-10  days  a  further  titration  is  made,  and  if  still  unsatisfactory  the 
animal  is  discarded.  As  a  rule,  three  animals  are  employed,  and  from  these 
at  least  one  will  produce  an  agglutinin  which  will  titrate  1-5000  or  higher. 
The  titer  may  be  maintained  by  subsequent  injections  at  longer  intervals,  but 
it  is  usually  found  desirable  to  kill  the  animal  by  bleeding  and  to  preserve  the 
serum  in  ampoules  in  the  refrigerator. 

Bleeding  the  Immune  Rabbit. — The  rabbit  may  be  "  bled  out  "  by  strapping 
it  on  a  flat  board,  lightly  anesthetizing  and  plucking  the  hair  from  the  groin 
on  one  side.  The  skin  is  scrubbed 
with  soap  and  water  and  then 
alcohol,  and  a  long  incision  made 
in  the  line  of  the  groin  groove. 
(See  Fig.  5.)  This  goes  through 
the  f ascias  and  exposes  the  femoral 
vessels.  The  neck  of  a  sterile 
150  c.c,  flask  is  placed  over  the 
vessels  just  below  Poupart's  liga- 
ment and  the  vessels  cut  with  the 
knife,  the  blood  being  caught  as 
it  spurts.  As  the  bleeding  con- 
tinues the  head  end  of  the  board 
is  raised  and  the  animal's  body 
squeezed  until  the  flow  ceases. 
Before  disposing  of  the  body, 
death  should  be  assured  by  a 
blow  fracturing  the  cervical  spine. 
The  flask  is  placed  in  an  oblique 
position  until  the  blood  is  firmly 
clotted,  then  placed  upright  in 
the  refrigerator.  (See  Fig.  6.) 
This  leaves  an  oblique  surface  of 
clot  from  which  the  serum  flows 
out  in  the  bottom  of  the  flask. 
The  blood  may  also  be  obtained 
from  the  carotid  artery,  but  this 
requires  more  careful  dissection, 
longer  anesthesia  and  may  require 
the  insertion  of  a  cannula.  Practi- 
cally it  gives  no  better  results 
either  in  quantity  of  blood  with- 
drawn or  sterility  of  the  process. 
Usually  15-20  c.c.  serum  are 
obtained  after  twenty-four  hours 
in  the  ice-chest,  and  only  occasion- 
ally is  it  necessary  to  centrifuge  in 
order  to  obtain  a  clear  serum.  The 
serum  is  withdrawn  as  shown  in 
Fig.  7  and  placed  in  small  sterile 
ampoules  of  dark  glass,  sealed  and 
kept  in  the  refrigerator. 

Macroscopic  Titration. — Titra- 
tion is  usually  by  the  macroscopic 
method,  but  an  alternative  is  the 
microscopic  method.  For  the  ti- 
tration by  the  macroscopic  method 
it  is  necessary  to  have  the  growth 
from  two  or  three  agar  slants, 
adding  about  10.0  c.c.  sterile  salt 

solution  to  each  tube  and  making  an  emulsion  as  described  for  immunization. 
The  suspension  is  placed  in  a  flask  and  killed  either  by  heat  (56°  C.-6o°  C.  for 
two  hours),  or  by  phenol  i.o  per  cent,  or  formalin  (40  per  cent.)  i.o  per  cent. 
The  killing  of  the  organisms  is  not  necessary,  but  is  desirable  because  of  the 
added  safety  and  because  such  killed  emulsions  may  be  preserved  in  the  refrigera- 
tor for  several  days  or  a  few  weeks.  Broth  cultures  may  be  used,  but  the 
hydrogen  ion  concentration  of  the  broth  may  add  a  small  factor  of  error  not 
present  in  the  saline  suspensions. 


PIG.  7.  —  Method  of  drawing  up  measured  volumes  into 
graduated   pipette.      The   rubber  tube  enables  the 


worker  to  observe  the  ascent  of  fluid  in  the  pipette 

and  the  position  of  the  tip  of  the  pipette.      Fluid  is 

withdrawn  from  flasks  in  the  same  fashion. 


84  THE  PRINCIPLES  OF  IMMUNOLOGY 

Dreyer,  who  has  given  much  attention  to  agglutination  in  the  diagnosis 
of  typhoid  and  paratyphoid  fevers  in  individuals  who  have  been  vaccinated 
against  these  diseases,  maintains  that  heat  and  chemicals  other  than  formal- 
dehyde are  inferior  to  the  latter  in  killing  and  preserving  the  bacterial  suspension. 
He  has  given  great  attention  to  standardization  of  the  reaction,  an  important 
but  not  infallible  precaution,  where  a  patient,  as  in  the  army,  is  likely 
to  be  examined  in  different  laboratories  during  the  course  of  the  disease. 
On  this  basis  Dreyer  has  shown  that  saline  emulsions  from  agar  cultures  are 
inferior  to  broth  cultures.  Scheimann  maintains  in  addition  that  the  broth 
cultures  furnish  a  more  permanent  standard.  Laboratories  in  which  such 
standards  are  prepared  determine  the  optimum  density  of  the  agglutinable 
cultures  and  also  keep  the  emulsions  until  the  early  deterioration  of  agglu- 
tinability  produced  by  the  formalin  has  reached  a  stationary  point,  after 
which  the  standards  remain  practically  unchanged  for  ten  months  and 
probably  longer. 

The  primary  test  is  carried  out  in  small  test  tubes,  with  each  dilution 
one-half  that  of  the  preceding  one.  This  simplifies  making  the  dilutions, 
especially  if  only  one  serum  is  to  be  tested.  A  row  of  twelve  tubes  is  placed 
in  a  rack  and  each  tube  receives  0.5  c.c.  salt  solution.  To  the  first  is  added 
0.5  c.c.  immune  serum,  the  mixture  blown  in  and  out  of  the  pipette  three  times 
and  0.5  c.c.  transferred  to  the  next  tube,  the  processes  repeated  and  0.5  c.c. 
transferred  to  the  next  tube,  and  so  until  the  last  tube  is  reached.  In  order 
to  preserve  the  constant  volume  in  each  tube,  0.5  c.c.  is  discarded  from  the 
last  tube.  Thus  there  are  dilutions  1-2,  1-4,  1-16,  1-32,  1-64,  1-128,  1-256,  1-512, 
1-1024,  1-2048,  1-4096.  To  each  tube  is  added  0.5  c.c.^bacillus  emulsion,  thus 
doubling  each  of  the  dilutions,  so  that  instead  of  ranging  from  1-2  to  1-4096, 
they  range  from  1-4  to  1-8192.  In  the  twelfth  tube  are  placed  0.5  c.c.  salt 
solution  and  0.5  c.c.  bacterial  emulsion  to  serve  as  a  control  of  the  emulsion 
and  prevent  error  due  to  spontaneous  clumping  of  the  organisms.  The  tubes 
are  placed  in  a  water  bath  at  37°  C.  for  one  hour  and  then  in  the  refrigerator 
over  night.  The  clumping  is  observed  with  the  naked  eye,  the  clumps  being 
visible  and  settling  more  rapidly  than  the  bacterial  emulsion.  Should  1-512 
of  the  final  dilution  show  agglutination  and  1-1024  fail  to^  show  it,  the  titer 
lies  between  these  two,  and  it  is  advisable  to  set  up  a  series  of  tubes  1-500, 
1-600,  1-800,  1-900,  i-iooo,  and  repeat.  The  same,  of  course,  is  true  of  the 
weaker  dilutions,  although  beyond  i-iooo  the  scale  is  more  easily  placed  in 
grades  of  200  rather  than  100.  The  preparation  of  such  dilutions  is  illustrated 
as  follows: 


0.5  c.c.  serum  +  4.5  c.c.  saline =  i-io    dilution 

0.5  c.c.  No.  i  +  12.0  c.c.  saline  =  1-25    dilution 

0.5  c.c.  No.  i  +  4.5  c.c.  saline  =  i-ioo  dilution 

0.5  c.c.  No.  2  -f  4.5  c.c.  saline  =  1-200  dilution 


4  0.5  c.c.  No.  2  -r  4.5  c.c.  saline - 

5  0.5  c.c.  No.  3  +  1.0  c.c.  saline  = 

6  0.5  c.c.  No.  2  +  5-5  c.c.  saline  == 

7  0.5  c.c.  No.  3  +  1.5  c.c.  saline  — 

8  0.5  c.c.  No.  2  +  8.5  c.c.  saline  — 

9  0.5  c.c.  No.  4  +  8.5  c.c.  saline  = 


-300  dilution 
-350  dilution 
-400  dilution 
-450  dilution 
-500  dilution 


Should  we  wish  to  determine  a  titer  between  1-200  and  1-500,  dilutions 
4-9  are  placed,  0.5  c.c.  in  each  of  six  tubes,  0.5  c.c.  emulsion  added  to  each, 
and  in  a  seventh  tube  0.5  c.c.  saline  and  0.5  c.c.  emulsion  as  a  control.  The 
tubes  are  placed  in  the  water  bath  and  incubated  as  before.  Similar  protocols 
may  be  made  if  higher  dilutions  are  required  for  the  final  test.  Some  workers 
prefer  to  set  up  primary  dilutions  of  i-io,  1-50,  i-ioo,  1-200,  1-500,  i-iooo, 
1-2000,  1-4000,  but  this  has  no  particular  advantages  as  compared  with  the 
primary  titration  outlined  above. 

Microscopic  Titration. — The  microscopic  method  may  be  employed  with 
the  same  method  of  dilution  and  mixing,  simply  removing  a  drop  for  obser- 
vation in  a  hanging  drop  preparation  at  the  end  of  the  period  of  incubation 
and  examining  with  a  4-mm.  lens.  Another  somewhat  less  accurate  method  is 
to  place  one  loopful  of  each  dilution  on  a  coverslip  and  mix  with  a  loopful  of 
bacterial  suspension,  inverting  the  slip  on  a  hollow  ground  slide,  sealing  with 
vaseline,  incubating  and  reading  the  result.  A  still  less  accurate  method  is 
to  place  on  coverslips  or  slides  a  row  of  loopfuls  of  salt  solution,  adding  a 
loopful  of  serum  to  the  first  drop,  mixing,  transferring  a  loopful  to  the  second 


FIG.   10. — Microscopic  drawing    showing    the  agglutina- 
tion of  a  suspension  of  bacillus  typhosus  by  blood  serum 
from  a  human   case   of   typhoid   fever,   as   seen   in   the 
Widal  test. 


AGGLUTININS  AND  PRECIPITINS 


drop  and  so  on  until  the  series  of  dilutions  has  been  made,  discarding  a  loop- 
ful  of  the  last  mixture  and  leaving  one  loopful  of  salt  solution  as  a  control, 
then  adding  to  each  drop  a  loopful  of  bacterial  suspension.  The  slips  or 
slides  are  inverted,  sealed,  incubated  and  read.  In  using  slides,  the  trouble 
of  sealing  may  be  avoided  by  incubating  in  a  moist  chamber.  The  micro- 
scopic method  is  usually  employed  in  the  Widal  test,  the  dilutions  of  patients' 
blood  or  serum  being  made  by  the  same  drop  method,  1-20,  1-40,  1-80.  Some- 
times a  drop  of  dried  blood  is  used,  this  being  laked  and  dissolved  by  a  drop 
of  water  and  then  made  up  to  the  first  dilution  of  1-20  by  the  addition  of 
nineteen  drops  of  saline.  Frequently  twenty-four-hour  broth  cultures  of  the 


FlG.  8. — The  Wright  tube  for  obtaining  small 
quantities  of  blood  serum. 


FIG.  p. — Coiled  pipette  for  taking  up  small  quantities  of  fluids.  Bubbling  in  the 
coil  gives  warning  of  the  filling  and  prevents  suction  into  the  mouth.  The  tube  may 
be  made  straight  and  plugged  with  C9tton.  Either  may  be  used  for  withdrawing 
serum  from  the  Wright  pipette.  Suction  may  be  applied  by  the  mouth  directly  or 
with  a  rubber  tube  or  by  means  of  a  small  nipple. 

typhoid  bacillus  are  employed  as  the  emulsion.  Clearer  results,  however,  are 
obtained  by  collecting  blood  in  Wright  tubes  (Fig.  8)  and  allowing  the 
serum  to  separate  for  dilution,  and  then  employing  a  salt  solution  suspension 
of  a  twenty-four-hour  agar  slant  culture. 

Specificity  of  Agglutinins — Group  Reactions. — >The  specificity 
of  the  reaction  may  be  shown  by  setting  up  dilutions  of  the  anti- 
typhoid serum  obtained  from  the  immunized  rabbit  against  suspen- 
sions of  bacillus  typhosus,  bacillus  paratyphosus  (A  or  B),  and 
bacillus  coli  communis.  An  illustrative  protocol  follows : 


Typhoid  immune  serum 

B.  typhosus 

B.  paratyphosus  A. 

B.  Coli 

1-4 

1-8 

+ 

+ 

+ 

-16 

-f 

+ 

+ 

-32 
-64 
-128 

| 

+ 

- 

-512 

+ 

— 

— 

-1024 
-2048 
-4096 

Salt  solution 

1 

— 

— 

This  protocol  illustrates  two  points,  first,  that  the  serum  agglu- 
tinates its  homologous  bacteria  in  high  dilutions,  and  second,  that 
in  strong  concentrations  it  also  agglutinates  other  organisms  of  the 
same  group.  Thus  the  specificity  is  not  absolute  throughout,  but 
there  is  a  "  zone  of  absolute  specificity,"  in  this  case  between  the 


86  THE  PRINCIPLES  OF  IMMUNOLOGY 

dilutions  of  1-128  to  1-4096.  The  fact  that  the  other  two  organisms 
are  agglutinated  is  due  to  the  phenomenon  of  "  group  reactions." 
In  the  same  way,  if  an  animal  were  immunized  to  bacillus  coli  the 
serum  would  agglutinate  coli  in  high  dilutions  and  typhosus  in 
lower  dilutions.  The  principle  is  also  well  shown  in  a  table  taken 
from  Citron : 

Typhoid  Cholera 

Agglutination  of  immune  immune 

serum  serum 

Against  B.  typhosus , , .  1-2,000  i-io 

Against  B.  paratyphosus i-ioo  i-io 

Against  B.  coli 1-25  i-io 

Against  V.  cholerae  i-io  1-3,000 

Absorption  of  Agglutinins. — If  specific  sera  for  paratyphosus  and 
coli  were  interposed  in  the  above  diagram  it  would  be  seen  that  these 
sera  would  clump  the  homologous  bacteria  in  high  dilution,  and  the 
others  of  the  group  in  only  low  dilutions.  This  indicates  that  in 
each  serum  we  may  assume  there  are  several  agglutinins,  one  for 
the  homologous  organism,  a  major  agglutinin  or  main  agglutinin, 
and  one  for  each  of  the  other  organisms  of  the  group,  minor  agglu- 
tinins or  partial  agglutinins.  This  statement  may  be  accepted  for 
the  present,  although  the  conception  will  be  somewhat  altered  in  the 
theoretical  discussion.  Castellani  has  shown  that  if  the  major 
agglutinin  is  absorbed  by  its  homologous  organism  the  minor  agglu- 
tinins disappear  also,  but  that  if  one  or  several  of  the  minor  agglu- 
tinins be  absorbed  by  other  members  of  the  group  of  organisms  the 
major  agglutinin  remains.  In  order  to  make  this  clear  we  shall  first 
illustrate  the  process  of  absorption  and  then  apply  it  to  the  group 
reaction.  It  is  well  known  that  an  animal  may  be  simultaneously 
immunized  to  two  or  more  types  of  organisms ;  for  example,  bacillus 
typhosus  and  bacillus  coli.  The  resulting  serum  may  agglutinate 
typhosus  in  dilution  of  1—4000  and  coli  in  dilution  of  i— 1000.  The 
absorption  of  the  agglutinins  may  be  shown  as  follows : 

Prepare  thick  suspensions  of  bacillus  typhosus  and  of  bacillus  coli  com- 
munis  by  suspending  the  twenty-four-hour  surface  growth  of  three  slant  agar 
cultures  in  about  5  c.c.  saline.  This  is  done  by  placing  5  c.c.  in  the  first  tube, 
making  the  suspension,  then  transferring  to  the  second  tube,  suspending  that 
culture  and  repeating  in  the  third  tube.  The  typhoid  emulsion  is  killed  by 
heat  of  56°  C.  for  one  hour  and  the  colon  by  heat  at  60°  C.  for  one  hour. 
Add  to  1.5  c.c.  serum  an  equal  volume  of  thick  suspension  of  dead  bacillus 
typhosus  and  in  another  tube  place  1.5  c.c.  serum  with  an  equal  volume  of 
thick  suspension  of  dead  colon  bacilli.  The  tubes  are  marked  A  and  B.  After 
mixing  the  emulsion  of  bacilli  and  serum  the  tubes  are  incubated  at  37°  C 
and  placed  in  the  ice-chest  for  twelve  hours.  The  tubes  are  centrifuged  and 
the  supernatant  fluid  pipetted  off.  The  bacteria  are  resuspended  and  the  sus- 
pensions diluted  with  salt  solution  about  1-20  or  more,  in  order  that  agglu- 
tination may  be  easily  observed.  The  supernatant  fluid  represents  a  1-2 
dilution  of  the  original  serum.  Place  0.5  c.c.  each  into  test  tubes  and  add  4.5  c.c. 
saline,  making  a  dilution  of  1-20,  well  under  the  titer  of  the  serum.  Of  the 
diluted  fluid  A  which  has  been  absorbed  by  typhosus  place  0.5  c.c.  in  a  series 
of  two  tubes  and  add  0.5  c.c.  thin  emulsion  of  colon.  After  incubation  the  first 
tube  will  show  no  agglutination,  and  the  second  tube  containing  colon,  whose 
agglutinin  has  not  been  absorbed,  will  show  agglutination.  Conversely  place 
0.5  c.c.  diluted  fluid  B  in  a  series  of  two  tubes,  and  add  in  order  0.5  c.c.  thin 


AGGLUTININS  AND  PRECIPITINS 


emulsion  of  typhosus  and  0.5  c.c.  thin  emulsion  colon.  After  incubation  only 
tube  i  shows  agglutination,  because  the  colon  agglutinins  have  been  ab- 
sorbed. The  protocol  of  this  experiment  with  the  controls  follows: 

SERIES  A  (ABSORBED  BY  TYPHOSUS) 

1.  Fluid  A  0.5  c.c.  4-  0.5  c.c.  typhosus  =  no  agglutination. 

2.  Fluid  A  0.5  c.c.  T  0.5  c.c.  colon  =  agglutination. 

SERIES  B  (ABSORBED  BY  COLON) 

3.  Fluid  B  0.5  c.c.  +  0.5  c.c.  typhosus  =  agglutination. 

4.  Fluid  B  0.5  c.c.  +  0.5  c.c.  colon  =  no  agglutination. 

CONTROLS 

5.  Saline  0.5  c.c.  +  0.5  c.c.  typhosus  —  no  agglutination. 

6.  Saline  0.5  c.c.  -f-  0.5  c.c.  color  =  no  agglutination. 

This  experiment  shows  only  the  essentials  of  the  specific  absorption.  It 
may  be  further  elaborated  by  making  a  series  of  dilutions  of  the  treated 
serum  so  as  to  show  the  fact  that  the  titer  is  essentially  unimpaired. 

Result 


Tube 

Fluid  A  0.5  c.c. 

Typhosus  emulsion 

I 

1-8 

0.5  c.c. 

2 

1-16 

0.5  c.c. 

3 

1-32 

0.5  c.c. 

4 

1-64 

0.5  c.c. 

1-128 

0.5  c.c. 

6 

1-256 

0.5  c.c. 

7 

1-512 

0.5  c.c. 

8 

1-1,024 

0.5  c.c. 

9 

1-2,048 

0.5  c.c. 

10 

1-4,096 

0.5  c.c. 

ii 

1-8 

Colon  emulsion 

12 

1-16 

0.5  c.c. 

13 

1-32 

0.5  c.c. 

14 

1-64 

0.5  c.c. 

15 

Saline 

0.5  c.c. 

16 

Saline 

Typhosus  0.5  c.c. 

At  the  same 

time  set  up  tubes 

as  follows: 

Tube 

Fluid  B  0.5  c.c. 

Colon  emulsion 

i 

1-8 

0.5  c.c. 

2 

1-16 

0.5  c.c. 

3 

1-32 

0.5  c.c. 

4 

1-64 

0.5  c.c. 

1-128 

0.5  c.c. 

6 

1-256 

0.5  c.c. 

7 

1-512 

0.5  c.c. 

8 

1-1,024 

0.5  c.c. 

9 

1-8 

Typhosus  emulsion 

10 

1-16 

0.5  c.c. 

ii 

1-32 

0.5  c.c. 

12 

1-64 

0.5  c.c. 

13 

Saline  0.5  c.c. 

0.5  c.c. 

14 

Saline  0.5  c.c. 

Colon  emulsion  0.5  c.c. 

Result 


This  experiment  shows  that  the  process  of  absorption  removes 
only  the  specific  agglutinin  and  leaves  the  other  agglutinin  un- 
changed. As  a  matter  of  practical  fact,  the  typhoid  agglutinin  re- 
mains unchanged,  but  the  colon  agglutinin  may  be  somewhat  reduced 
in  titer,  perhaps  to  1-800  or  even  as  low  as  1-300.  In  a  combined 
serum  of  this  sort  with  the  typhoid  agglutinin  of  high  titer,  part  of 
the  agglutinin  for  colon  is  the  result  of  a  typhoid  minor  agglutinin 
which  is  removed  by  absorption  with  typhosus,  thus  reducing  the 


88 


THE  PRINCIPLES  OF  IMMUNOLOGY 


colon  titer.  The  primary  colon  titer  of  1000  would  have  a  very  low 
content  of  minor  agglutinin  for  typhosus,  the  removal  of  which 
would  leave  the  primary  titer  for  typhosus  practically  unchanged 
after  absorption  with  colon  bacilli. 

The  differences  of  absorption  of  major  and  minor  agglutinins  may  be 
illustrated  by  the  use  of  a  typhosus  immune  serum.  We  may  use,  for  illustra- 
tion, as  closely  related  organisms  bacillus  typhosus  and  bacillus  paratyphosus  B. 
Preliminary  titration  of  the  serum  is  carried  out  as  usual  against  bacillus 
typhosus  and  bacillus  paratyphosus  B.  Let  us  suppose  that  the  serum  shows 
a  titer  of  1-4096  for  typhosus  and  1-512  for  paratyphosus  B.  Thick  emulsions 
of  typhosus  and  para  B  are  made  as  described  in  the  previous  experiment, 
killed  by  56°  C.  for  one  hour  and  mixed  in  equal  volume  with  1.5  c.c.  serum, 
incubated  for  one  hour  and  refrigerated  for  twelve  hours,  then  centrifuged 
and  the  fluid  pipetted  off.  The  experiment  with  the  results  may  be  illus- 
trated in  the  following  protocol: 


Tube 

I 
2 

3 
4 


Fluid  A  0.5  c.c. 

(absorbed 
by  typhosus) 

-16 


-128 

-256 

-512 

-1,024 

-2,048 


Typhosus  emulsion 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 


Fluid  A  0.5  c.c. 

Tube 

(absorbed  by 

Para  B  emulsion 

typhosus) 

10 

1-16 

0-5 

II 

1-32 

0-5 

12 

1-64 

0-5 

13 

1-128 

0.5 

14 

1-256 

'0-5 

15 

16 

Saline  0.5  c.c. 
Saline  0.5  c.c. 

Typhosus  0.5  c.c. 
Para  B.  0.5  c.c. 

Fluid  B  0.5  c.c. 

Tube 

(absorbed  by 

Typhosus  emulsion 

Para  B) 

11 

-16 

-32 

0.5  c.c. 
0.5  c.c. 

19 

-64 

0.5  c.c. 

2O 

-128 

0.5  c.c. 

21 

-256 

0.5  c.c. 

22 

-512 

0.5  c.c. 

23 

-1,024 

0.5  c.c. 

24 

-2,048 

0.5  c.c. 

25 

-4,096 

0.5  c.c. 

Para  B  emulsion 

26 

-16 

0.5  c.c. 

27 

-32 

0.5  c.c. 

28 

-64 

0.5  c.c. 

29 

-128 

0.5  c.c. 

30 

-256 

0.5  c.c. 

3i 

Saline  0.5  c.c. 

0.5  c.c. 

32 

Saline  0.5  c.c. 

Typhosus  0.5  c.c. 

Untreated  serum 

33 

1-2,048 

Typhosus  0.5  c.c. 

34 

1-256 

Para  B.  0.5  c.c. 

Result 


Result 


Result 


AGGLUTININS  AND  PRECIPITINS  89 

It  will  be  seen  from  these  protocols  that  absorption  by  the  major  agglu- 
tinogen,  bacillus  typhosus,  removes  both  the  major  and  minor  agglutinins, 
and  that  absorption  by  the  minor  agglutinogen  removes  only  the  minor  agglu- 
tinin,  although  it  is  true  that  even  though  the  titer  of  the  major  agglutinin  is 
not  reduced  it  may  agglutinate  in  smaller  clumps. 

Inhibition  Zones. — It  is  sometimes  found  that  in  powerful  agglu- 
tinins there  is  an  "  inhibition  zone  "  in  the  more  concentrated  dilu- 
tions. Thus  a  serum  may  agglutinate  as  follows : 

Tube  Serum  dilution  Result 

^  I  I-IO 

2  I-IOO  + 

3  1-1,000  +-H- 

4  1-2,000  -f-H- 

5  1-4,000  -}— f- 

6  1-6,000  + 

7  1-8,000 

This  phenomenon  is  somewhat  more  frequently  observed  in  sera 
that  have  been  preserved  for  a  considerable  time  in  the  moist  state. 
If  a  serum  with  a  titer  of  i-iooo,  which  originally  showed  agglu- 
tination in  all  dilutions  up  to  1000,  is  preserved  and  after  several 
months  titrated  again,  it  may  fail  to  agglutinate  in  i-io,  may 
agglutinate  only  weakly  in  i-ioo,  and  completely  in  1-500.  If  the 
tube  containing  i-io  dilution  is  centrifuged,  the  supernatant  fluid 
drawn  off,  the  bacteria  again  suspended  and  placed  with  the  serum 
in  dilution  of  1-500,  there  is  no  agglutination.  The  same  is  true  if 
these  treated  organisms  are  placed  in  contact  with  a  fresh  agglu- 
tinating serum.  The  same  phenomenon  is  obtained  if  the  serum 
first  used  is  a  fresh  one  of  high  titer  with  an  inhibition  zone, 
and  the  bacteria  are  removed  from  the  low  dilutions  in  which  they 
have  failed  to  agglutinate.  The  bacteria  have  become  inagglutin- 
able  by  treatment  with  the  serum  in  these  concentrations.  Simi- 
larly, heating  an  agglutinating  serum  to  65°  to  70°  C.  destroys  its 
agglutinating  properties,  but  if  it  is  added  to  bacteria  they  become 
inagglutinable  when  treated  with  fresh  active  serum.  This  phe- 
nomenon is  strictly  specific  and  operates  only  in  the  presence  of  the 
homologous  organism.  This  peculiar  character  of  agglutinins  has 
been  closely  linked  with  the  Ehrlich  conception  of  immune  bodies 
and  is  explained  as  due  to  the  presence  in  sufficient  concentration  of 
"  agglutinoids."  The  term  agglutinoid  is  applied  to  that  part  of  the 
agglutinin  which  has  a  specific  binding  affinity  for  the  cell,  but  has 
been  deprived  of  the  thermolabile  and  more  easily  destructible  frac- 
tion which  has  the  power  of  producing  clumping.  This  explanation 
will  be  discussed  more  in  detail  in  the  general  discussion 
of  agglutinins. 

The  influence  of  heat  on  agglutination  has  been  studied  exten- 
sively. As  has  been  indicated,  heat  will  destroy  agglutinins,  but 
certain  agglutinins  are  destroyed  by  degrees  of  heat  which  fail  to 
destroy  others.  Most  agglutinating  sera  are  rendered  inactive  at 
60°  to  65°  C.,  but  anti-plague  agglutinin  is  destroyed  at  56°  C, 


90  THE  PRINCIPLES  OF  IMMUNOLOGY    f> 

whereas  others  do  not  disappear  until  80°  C.  has  been  reached. 
Wells  states  that  "  purified  typhoid  agglutinin  may  resist  80  to  90 
degrees."  Agglutinins  cannot  be  reactivated  by  the  addition  of 
fresh  serum,  even  though  the  temperature  may  have  been  adjusted 
so  that  the  agglutinoid  remains. 

A  simple  experiment  for  the  demonstration  of  the  influence  of  heat  on 
agglutinins  is  as  follows:  The  typhoid  immune  serum,  the  production  of 
which  has  been  described  above,  and  also  the  killed  typhoid  suspension  may 
be  used.  In  each  of  three  tubes  place  0.5  c.c.  serum  diluted  i-io,  and  into  a 
fourth  tube  0.5  c.c.  salt  solution.  Tube  I  is  heated  in  a  water  bath  at  56°  C. 
for  one-half  hour,  tube  2  heated  at  70°  to  75°  C.  for  one-half  hour,  and 
tubes  3  and  4  kept  at  room  temperature.  After  cooling  tubes  i  and  2,  add 
0.5  c.c.  bacterial  emulsion  to  each  tube  and  incubate  for  one  hour  at  37°  C. 
Agglutination  will  not  occur  in  tube  2,  the  serum  having  been  heated  to  70°  to 
75°  C.,  nor  in  the  control  tube  with  saline.  The  unheated  serum  and  the 
serum  heated  to  56°  C.  will  agglutinate  powerfully.  It  will  be  found  also  that 
the  addition  of  o.i  c.c.  fresh  guinea-pig  serum  (complement)  to  tube  2,  and 
subsequent  incubation  will  fail  to  produce  agglutination. 

It  is  of  interest  to  note  that  the  degree  of  concentration  of  serum 
has  some  influence  on  the  degree  of  heat  necessary  for  destruction. 
For  example,  Koeckert  in  this  laboratory  found  that  normal  un- 
diluted iso-hemagglutinins  are  destroyed  at  65°  to  66°  C.  for 
thirty  minutes,  but  that  in  high  dilutions  they  are  destroyed  at  62°  C. 
for  thirty  minutes. 

The  influence  of  electrolytes  on  the  phenomenon  of  agglutination 
is  of  considerable  importance  from  the  theoretical  point  of  view 
because  of  the  resemblance  to  flocculation  of  colloidal  suspensions. 
Bordet,  who  discovered  this  fact,  compared  the  reaction  to  the 
throwing  down  of  the  alluvial  matter  in  rivers  as  the  fresh  water 
meets  the  salt  water  of  the  ocean.  By  previously  dialyzing  the  salts 
out  of  the  bacterial  suspension  and  the  specific  serum  he  showed 
that  agglutination  would  not  occur,  but  that  if  the  mixture  was 
salted  in  proper  concentration  the  reaction  would  take  place.  It  is 
possible,  however,  to  agglutinate  bacteria  by  certain  concentration 
of  salts,  particularly  of  the  heavy  metals,  but  such  concentration  is 
always  much  stronger  than  is  necessary  for  salting,  as  described  in 
the  Bordet  experiment. 

The  demonstration  of  the  influence  of  salts  may  be  seen  in  the  following 
experiment,  taken  from  Zinsser,  Hopkins  and  Ottenberg.  For  this  experi- 
ment the  killed  typhoid  suspension  and  the  anti-typhoid  serum  as  employed 
in  previous  experiments  may  be  used.  "  Place  in  each  of  two  centrifuge  tubes 
with  pointed  tip  2.0  c.c.  of  the  suspension.  To  tube  A  add  2.0  c.c.  of  agglu- 
tinating serum  diluted  1-50.  To  tube  B  add  2.0  c.c.  distilled  water.  Allow 
the  tubes  to  stand  at  37°  C.  for  thirty  minutes.  Centrifugalize  the  tubes  at 
high  speed  until  the  supernatant  fluid  is  clear."  Pipette  off  the  fluid  and  "  to 
the  washed  sediments  add  2.0  c.c.  distilled  water  and  draw  the  mixture  re- 
peatedly in  and  out  of  the  capillary  pipette  in  order  to  break  up  the  clumps  and 
obtain  an  even  suspension.  Set  up  the  following  tests  in  agglutination  tubes : 

1  Sediment  A  0.5  c.c Distilled  water  0.5  c.c. 

2  Sediment  A  0.5  c.c.        10  per  cent  NaCl  0.09  c.c.     Distilled  water  0.5  c.c. 

•  3  Sediment  A  0.5  c.c.       0.8  per  cent.  CuSO4  0.02  c.c.  Distilled  water  0.5  c.c. 
4  Sediment  B  0.5  c.c.       0.8  per  cent.  CuSO4  0.02  c.c.  Distilled  water  0.5  c.c. 

•  5  Sediment  B  0.5  c.c.       0.8  per  cent.  CuSO4  o.i    c.c.  Distilled  water  0.5  c.c. 
6  Sediment  B  0.5  c.c Distilled  water  0.5  c.c. 


AGGLUTININS  AND  PRECIPITINS  91 

"The  tubes  are  placed  in  the  water  bath  at  37°  C  for  one  hour  and  then 
observed.  Tubes  2,  3  and  5  should  show  agglutination."  In  tube  A  the 
bacteria  have  been  '  sensitized  '  with  the  immune  serum,  and  after  the  clumps 
have  been  broken  up  are  ready  again  for  clumping  under  proper  conditions. 
In  tube  i  the  addition  of  distilled  water  does  not  provide  the  essential  con- 
ditions, but  in  tubes  2  and  3  the  addition  of  electrolytes  favors  the  reaction. 
In  tube  B  the  bacteria  have  not  been  sensitized,  but  of  the  tubes  4,  5  and  6, 
the  concentration  of  the  copper  sulphate  is  such  as  to  induce  clumping  in 
itself,  a  phenomenon  frequently  seen  in  certain  concentrations  of  salts  of 
the  heavy  metals,  such  as  zinc,  lead  and  mercury. 

Influence  of  Hydrogen  Ion  Concentration. — It  has  been  shown 
by  Michaelis  and  others  that  bacteria  may  be  agglutinated  by  pro- 
viding a  proper  hydrogen  ion  concentration,  and  it  was  hoped  that 
this  might  provide  a  means  of  rapid  identification  of  organisms. 
Proteins,  for  example,  have  a  specific  and  constant  optimum  con- 
centration of  H  ions  for  their  precipitation.  In  the  case  of  bacteria 
it  was  shown,  for  example,  that  bacillus  typhosus  was  agglutinated 
by  a  hydrogen  ion  concentration  of  4  to  8  X  io~5,  whereas  para- 
typhosus  requires  16  to  32  X  icr5,  colon  bacilli  not  being  agglutin- 
able  by  this  method.  It  has  been  shown,  however,  that  this 
differentiation  is  not  so  sharp  as  was  at  first  supposed,  that  differ- 
ent strains  show  considerable  irregularity,  and  that  there  is  over- 
lapping of  one  species  with  another.  A  combination  of  serum  and 
acid  agglutination  has  shown  that  bacteria  sensitized  by  serum  can 
be  more  readily  agglutinated  than  are  non-sensitized  bacteria.  The 
specific  characters  of  bacterial  proteins  are  probably  due  to  such  a 
slight  variation  in  the  arrangement  of  the  molecular  structure  that 
a  satisfactory  differentiation  by  changes  in  hydrogen  ion  concentra- 
tion is  not  at  present  feasible.  Eisenberg  has  recently  studied  the 
problem  with  584  races  of  bacteria,  of  which  537  were  of  the  colon- 
typhoid  group,  and  found  no  differential  diagnosis  possible  with  the 
acid  agglutination  method.  He  also  found  flocculation  with  salts 
of  the  heavy  metals  extremely  variable. 

The  Mechanism  of  Agglutination. — The  data  given  in  the  pre- 
ceding paragraphs  outline  the  most  important  phases  of  the  phe- 
nomenon of  agglutination,  and  any  discussion  of  the  mechanism  of 
the  process  must  be  based  on  these  fundamentals.  The  chemical 
nature  of  the  agglutinogen  is,  of  course,  closely  combined,  if  not 
identical,  with  the  protein  of  the  cells,  but  is  in  no  sense  dependent 
for  its  activity  on  the  existence  of  life  within  the  cell.  Agglutinogens 
are  not  destroyed  by  mild  concentrations  of  formalin,  phenol,  heat, 
or  ultra-violet  rays  which  are  sufficient  to  destroy  the  life  of  the  cell 
itself.  They  pass  through  dialyzing  membranes  more  rapidly  than 
do  the  agglutinins,  and  therefore  are  probably  made  up  of  smaller 
molecules.  That  they  pass  through  collodion  sacs  can  be  shown  by 
implanting  such  sacs,  filled  with  killed  typhoid  organisms,  in  the 
peritoneal  cavity  of  rabbits  and  observing  the  development  of  agglu- 
tinins in  their  blood;  an  observation  which  has  been  confirmed  by 
Reimann  in  this  laboratory.  Old  broth  cultures  contain  in  the  fluid 
agglutinogens  which  may  neutralize  agglutinins  and  which  may 


92  THE  PRINCIPLES  OF  IMMUNOLOGY 

serve  also  to  produce  agglutinins  upon  injection.  Thus  it  would 
appear  that  agglutinogens  are  bodies  of  small  molecular  size  capable 
of  slow  diffusion  and  almost  certainly  protein,  although  Stuber 
maintains  that  they  are  of  fatty  nature.  The  influence  of  heat  on 
agglutinogens  has  been  carefully  studied  by  Joos,  who  concluded 
that  the  agglutinogen  consists  of  relatively  thermolabile  and  ther- 
mostable constituents  (the  dividing  line  being  60°  to  62°  C.)  which 
induce  the  formation  of  separate  agglutinins.  The  thermostable 
fraction  resists  heat  up  to  165°  C.,  is  soluble  in  alcohol,  and  does 
not  give  protein  reactions,  whilst  the  thermolabile  fraction  gives  all 
the  protein  reactions.  This  work  is  more  fully  discussed  subsequently. 

Alterations  of  Agglutinability. — Of  considerable  interest  in  con- 
nection with  agglutinogens  is  the  alteration  of  agglutinability  of  the 
cell.  This  probably  is  more  closely  associated  with  the  cell  as  such 
than  with  the  agglutinogen.  If  bacteria  are  heated  above  65°  C. 
they  are  not  agglutinable  by  specific  immune  sera,  but  can  absorb 
agglutinin  from  the  sera.  Organisms  freshly  isolated  from  cases  of 
infectious  disease  often  show  similar  reductions  of  agglutinability, 
but  recover  it  after  prolonged  growth  on  artificial  media.  This  is 
likely  to  be  true  in  the  case  of  "  carriers,"  and  Welch  has  referred  to 
it  as  a  quasi-immunity  which  the  bacteria  themselves  have  acquired 
by  acting  against  the  immune  bodies  of  the  host,  an  immunity,  how- 
ever, which  the  organisms  lose  on  living  in  the  environment  of  the 
artificial  culture  media.  Such  inagglutinability  may  be  produced 
artificially  by  growing  the  bacteria  on  media  containing  a  specific 
immune  serum,  heated  to  destroy  any  bacteriolytic  influence.  In  a 
personal  communication  to  us  M.  Cooper  has  stated  that  the  pres- 
ence of  capsules  about  bacteria  serves  to  establish  a  quasi-immunity 
for  the  organisms  against  antibodies,  and  that  such  capsules  appear 
after  cultivation  in  immune  sera.  This  peculiar  phenomenon  is  ex- 
plained on  the  Ehrlich  theory  by  assuming  that  the  bacteria  are 
practically  exhausted  of  receptors.  Nevertheless,  such  inagglu- 
tinable  bacteria  upon  injection  into  animals  lead  to  the  production 
of  agglutinins  for  agglutinable  strains,  but  not  for  inagglutinable 
strains.  It  has  also  been  assumed  that  they  are  saturated  with 
agglutinoid,  but  in  America,  at  least,  the  Welch  theory  has  been 
given  wide  acceptance  as  an  important  philosophical  conception. 
Not  only  may  agglutinability  be  altered,  but  different  strains  of  an 
organism  show  natural  differences  in  agglutinability.  For  example, 
Cole  has  shown  that  against  a  specific  agglutinating  serum  five 
strains  of  pneumococcus  showed  titers  of  1-4000,  1-4500  (2),  1-7000, 
and  1-8000.  These  are  not  "  types  "  of  a  species  but  strains,  and  show 
no  specific  agglutinability  for  sera  produced  by  the  strain  in  question. 

The  Nature  of  Agglutinins. — The  chemical  study  of  the  agglu- 
tinins shows  that,  like  antitoxins,  they  are  precipitated  out  of  the 
serum  in  the  globulin  fraction,  and  so  far  they  have  not  been  fur- 
ther purified.  They  pass  through  filters  less  readily  than  their 
antigens,  and  therefore  have  a  larger  molecular  structure.  Pepsin 


AGGLUTININS  AND  PRECIPITINS  93 

digestion  destroys  the  agglutinins  fairly  readily,  but  trypsin  acts 
more  slowly.  Alkalies  even  when  dilute  are  destructive,  but  acids 
operate  less  actively.  They  are  absorbed  by  charcoal.  They  are 
not  thrown  down  in  the  precipitate  formed  by  specific  precipitating 
sera.  The  influence  of  heat  on  agglutinins  has  been  the  subject  of 
much  study.  The  work  of  Joos  was  conducted  with  both  agglu- 
tinogen  and  agglutinin.  As  mentioned  above,  he  demonstrated  the 
presence  in  the  bacterial  antigen  of  a  thermolabile  A  agglutinogen 
and  a  thermostable  B  agglutinogen,  the  dividing  line  being  60°  to 
62°  C.  The  injection  of  heated  antigen  (B  agglutinogen)  gives  rise 
to  the  formation  of  B  agglutinin,  which  in  contrast  to  the  antigen  is 
destroyed  by  heat  of  60°  C.,  but  reacts  with  both  A  and  B  agglu- 
tinogens.  The  injection  of  the  unheated  bacilli  containing  both  A 
and  B  agglutinogen  leads  to  formation  of  both  agglutinins,  but  the 
B  agglutinin  can  be  removed  by  heat  leaving  the  thermostable  A 
agglutinin,  which  reacts  only  with  the  A  agglutinogen.  The  essen- 
tials of  this  work  have  been  confirmed,  although  Scheller  working 
with  bacillus  typhosus  found  that  the  B  agglutinin  is  reduced  in  titer 
but  not  completely  destroyed  at  60°  to  62°  C.  Scheller  showed 
further  that  the  heated  bacteria  (B  agglutinogen)  absorb  agglu- 
tinins from  the  sera  more  readily  than  do  unheated  bacteria,  and  that 
they  give  the  highest  titers  with  the  serum. 

According  to  the  Ehrlich  scheme,  agglutinins  have  a  haptophore  or 
combining  group  and  a  zymophore  group  which  causes  the  agglutination. 
This  zymophore  is  killed  by  heat  and  deteriorates  on  long  standing  to  form 
the  agglutinoid  (or  agglutinin  free  from  zymophore),  which  has  combin- 
ing but  not  agglutinating  power.  Thus  in  the  side-chain  theory  the 
agglutinins  (and  precipitins)  differ  from  the  theoretical  simplicity  of 
the  antitoxins  and  constitute  the  receptors  of  the  second  order. 

The  Physical  Basis  of  Agglutination. — The  mechanism  of  agglu- 
tination is  such  that  the  reaction  takes  place  in  constant  proportions, 
thus  likening  it  to  a  simple  chemical  reaction.  The  reaction  is  re- 
versible, however,  in  that  simple  shaking,  the  use  of  organic  and 
inorganic  acids  and  acid  salts,  as  well  as  alkalies  and  heat  of  70°  to 
75°  C.,  can  break  the  clumps  into  cell  units;  but  after  this  separa- 
tion fresh  agglutinating  serum  cannot  operate  again.  It  has  been 
shown  further  that  agglutinins  can  be  separated  from  bacteria- 
agglutinin  combinations  by  the  electric  current;  therefore,  the  agglu- 
tinins are  not  destroyed  by  the  union  with  the  bacteria.  Many  of 
the  older  workers  believed  that  the  reaction  occurred  because  of 
changes  in  the  outer  layers  or  ectoplasmic  substance  of  the  cells. 
Gruber  at  first  maintained  that  a  substance,  glabrificin,  was  taken 
from  the  serum  by  the  cejls  which  made  their  outer  surfaces  sticky 
and  caused  adhesions  when  their  motility  brought  the  bacteria  in 
contact  with  one  another.  Malvoz  and  others  held  that  the  reaction 
depended  upon  the  entanglement  of  the  flagella  of  the  bacteria. 
Neither  of  these  ideas  is  consistent  with  the  fact  that  non-motile 


94  THE  PRINCIPLES  OF  IMMUNOLOGY 

bacteria  and  other  cells  are  subject  to  agglutination,  but  no  definite 
proof  is  at  hand  to  show  that  the  ectoplasmic  substance  is  not  of 
considerable  importance.  The  influence  of  salts  on  agglutination 
lends  much  support  to  the  conception  that  agglutination  is  a  col- 
loidal phenomenon.  As  has  been  indicated  above,  the  presence  of 
electrolytes  is  essential  to  the  reaction,  but  salts,  acids,  and  salts  of  heavy 
metals,  if  present  in  sufficient  concentration,  may  of  themselves  produce 
agglutination.  On  the  other  hand,  salts  in  strong  concentration 
serve  to  prevent  the  action  of  agglutinin.  When  bacteria  have  ab- 
sorbed agglutinin,  very  small  amounts  of  salt  serve  to  bring  about 
agglutination.  If  a  suspension  of  bacteria  and  an  agglutinating 
serum  are  each  dialyzed  free  of  salt  and  the  two  mixed,  the  bacteria 
absorb  agglutinin.  This  is  shown  by  the  fact  that  the  supernatant 
fluid  after  centrifugalization  is  free  of  agglutinin,  but  agglutination 
occurs  on  addition  of  salt.  Bordet  interpreted  the  phenomenon  of 
agglutination  as  having  two  phases,  first  that  of  sensitization  of  the 
bacteria  by  the  agglutinin,  and  second,  that  of  agglutination  of  these 
agglutinin-bacteria  by  the  salt.  It  may  be  stated  in  other  terms 
that  the  bacteria  are  primarily  suspensions  of  protected  colloids 
which  are  so  altered  by  the  agglutinin  that  they  become  unprotected 
and  precipitable  by  salts,  or  that  they  become  more  permeable  for 
electrolytes.  In  fact,  it  has  been  shown  that  sensitized  bacteria 
take  up  salts  more  readily  than  unsensitized.  The  similarity  of 
bacteria  to  protected  colloids  is  also  borne  out  by  Porges,  who 
showed  that  while  encapsulated  organisms  are  inagglutinable,  the 
solution  of  their  capsules  by  heating  in  weak  acid  renders  the  bac- 
teria agglutinable.  Bacteria  carry  electro-negative  charges  and  move 
toward  the  anode,  whereas  agglutinins  are  electro-positive.  The 
sensitized  bacteria  are  agglutinated  by  the  current  between  the  poles, 
although  the  sensitized  bacteria  move  slowly  toward  the  anode. 
The  small  amount  of  salt  necessary  for  agglutination  further  sup- 
ports the  influence  of  electrical  charge  and  thus  furnishes  further 
analogy  with  colloidal  precipitation.  Neisser  and  Friedemann  have 
studied  the  similarities  of  agglutination  and  colloidal  precipita- 
tion and  offer  much  in  support  of  such  analogy.  Two  protocols 
may  serve  to  show  the  importance  of  their  work,  one  dealing 
with  the  so-called  sensitization  and  the  other  with  inhibition  zones. 
Just  as  salt  influences  agglutinin  and  agglutinogen,  so  may  it 
influence  mastic  and  gelatin  solutions,  as  may  be  seen  in  the  follow- 
ing experiment : 

i.o  c.c.  mastic  i.o  c.c.  mastic  +  o.oooi  c.c. 
(i-io  original  emulsion)  2%  gelatin  sol.  and 

10%  NaCl  Sol.  diluted  to  3.0  c.c.  diluted  to  3.0  c.c. 

I.O        C.C.  +  + 

0.5  c.c.  +    . 

0.25  c.c. 

0.125  c.c.  + 

0.05  c-c. 

0.025  c.c.  —  — 


AGGLUTININS  AND  PRECIPITINS  95 

Furthermore,  they  offer  a  protocol  showing  the  similarity  be- 
tween the  reaction  of  colloidal  iron  hydroxide  upon  mastic  emul- 
sions and  the  agglutination  phenomenon  in  reference  to  inhibition 
zones.  It  will  be  seen  that  stronger  concentrations  of  the  iron 
hydroxide  fail  to  precipitate,  thus  simulating  the  action  of  strong 
concentrations  of  an  agglutinating  serum  of  high  titer  or  of  an  old 
serum.  The  protocol  follows  : 

Colloidal  iron  hydroxide         Mastic  emulsion  Result 

I.O  .0    C.C. 

0.5  .o  c.c. 

0.25  .o  c.c. 

O.I  .O   C.C. 

0.5  .o  c.c. 

O.O25  2.O    C.C. 

O.OI  .0   C.C. 

0.005  .0  c.c. 

0.0025  .o  c.c. 

O.OOI  .O    C.C. 

This  latter  protocol  is  of  significance  not  only  in  relation  to 
agglutination,  but  is  of  importance  also  in  connection  with  the 
Neisser-Wechsberg  phenomenon  of  complement-deviation  (not 
fixation)  discussed  in  connection  with  bacteriolysis.  As  Zinsser  says, 
"  it  seems  to  be  a  universal  fact  governing  the  union  of  colloidal 
substances,  that  definite  quantitative  proportions  must  be  main- 
tained in  order  to  lead  to  reaction,  this  being,  possibly,  explicable  on 
the  basis  that  actual  union  can  take  place  only  after  disturbance  of 
the  electrical  balance  which  keeps  the  particles  apart."  The  assump- 
tion that  agglutinoids  have  an  important  bearing  on  the  presence  of 
inhibition  zones  is  not  necessary  if  we  accept  the  colloidal  nature  of 
agglutination.  This  does  not  entirely  controvert  the  existence  of 
altered  agglutinin  with  a  binding  power  for  agglutinogen. 

Not  only  may  salt-free  bacteria-agglutinin  combinations  be 
agglutinated  by  salts  but,  as  Friedberger  has  shown,  certain  organic 
substances,  such  as  dextrose  and  asparagin,  serve  also  to  produce 
agglutination  in  such  salt-free  mixtures.  These  substances  do  not 
dissociate  in  solution  as  do  salts,  and  therefore  produce  no  electric 
phenomena.  This  fact  presents  a  certain  objection  to  the  final 
acceptance  of  the  colloidal  theory  of  agglutination,  but  it  is  possible 
that  the  mechanism  in  this  instance  is  of  a  nature  different  from 
that  of  the  immunological  process,  and  certainly  the  great  mass  of 
evidence  is  in  favor  of  the  reaction  of  agglutination  being  of 
colloidal  nature. 

Nothing  has  been  definitely  brought  forward  in  the  physico- 
chemical  examination  of  agglutination  to  explain  specificity,  except 
the  fact  previously  indicated,  that  variations  of  hydrogen  ion  con- 
centration have  a  relatively  specific  action  on  bacteria.  As  is 
known,  the  definite  identification  of  bacteria  by  this  method  has 
not  been  satisfactory.  The  specificity  of  immune  serum  agglutina- 


96 


THE  PRINCIPLES  OF  IMMUNOLOGY 


FIG.  ii.— The  nip- 
ple pipette  for 
making  mixtures 
of  fluids  and  bac- 
teria] suspension. 
The  pencil  mark  is 
seen  a  short  dis- 
tance above  the 
tip. 

Tube 

I 
2 

3 

4 
5 


tion  is  also  a  relative  matter,  as  is  shown  in  the  group 
reactions,  and  if  electric  phenomena  play  a  part  in  spe- 
cificity they  are  more  delicate  than  can  be  demon- 
strated by  present  chemical  or  electrical  methods. 
Bordet,  who  laid  no  emphasis  on  the  electrical  reac- 
tions, thought  that  the  process  of  sensitization  of  bac- 
teria by  agglutinins  is  in  essence  a  denaturing  of  the 
bacterial  proteins,  and  that  the  specificity  of  the  process 
depends  on  the  degree  of  denaturation. 

The  Dreyer  Test.— The  Widal  test  has  been  described 
(page  85).  This  test  has  been  of  the  greatest  service  in 
the  diagnosis  of  typhoid  and  paratyphoid  fevers  but  the 
introduction  of  vaccination  on  a  large  scale  has  reduced  the 
value  of  the  test  as  a  diagnostic  sign  of  actual  disease,  be- 
cause vaccinated  individuals  give  a  positive  test.  Dreyer 
studied  the  course  of  agglutination  in  typhoid  and  paratyphoid 
fevers,  and  found  that  the  agglutinative  titer  of  the  blood 
follows,  during  the  course  of  the  disease,  a  fairly  regular  curve, 
increasing  to  the  third  week  and  then  declining.  Although 
the  titer  may  be  higher  at  the  beginning  of  the  disease  in 
vaccinated  individuals  than  in  others,  the  titer  follows  the 
same  general  curve.  Of  more  importance  is  the  differentia- 
tion between  typhoid  and  other  infections  in  the  vaccinated. 
This  has  been  of  the  utmost  importance  in  the  World  War 
in  distinguishing  between  febrile  disease,  such  as  trench  fever 
or  malaria,  and  typhoid  or  paratyphoid.  The  test  is  made  by 
the  macroscopic  method  for  agglutination,  and  must  be  re- 
peated at  weekly  intervals  in  order  to  determine  the  curve 
of  agglutinins.  Not  infrequently  the  first  test  may  show  a 
titer  so  much  higher  than  occurs  after  vaccination  that  a 
presumptive  diagnosis  is  justifiable.  Under  war  conditions 
the  transfer  of  patients  often  made  it  necessary  to  perform 
the  tests  in  several  different  laboratories,  and  to  provide  for 
this  the  Oxford  Standards  Laboratory  prepared  emul- 
sions of  the  bacilli  for  distribution.  For  this  purpose 
the  organisms  were  grown  for  twenty-four  hours  in  pep- 
ton  veal  broth,  then  shaken  well  and  o.i  per  cent,  for- 
malin (40  per  cent,  formaldehyde)  added.  The  culture  was 
stored  at  2°  C.  and  shaken  frequently  during  four  or 
five  days.  At  the  end  of  this  time  it  was  usually  sterile. 
It  was  then  diluted  to  standard  opacity  by  means  of  salt 
solution,  to  which  was  previously  added  o.i  per  cent, 
formalin.  It  was  further  standardized  as  to  ^agglutina- 
bility  and  labeled  with  a  factor  so  as  to  provide  means 
whereby  tests  in  different  laboratories  could  be  estimated 
on  the  same  basis. 

The  blood  for  the  test  can  be  obtained  in  a  Wright 
tube,  but  it  is  preferably  taken  from  the  cubital  vein 
into  a  centrifuge  tube,  so  as  to  provide  a  fairly  large 
amount  of  serum.  In  order  to  make  the  method  appli- 
cable in  laboratories  where  graduated  pipettes  are  ^  not 
available,  Dreyer  made  all  the  dilutions  with  a  nipple 
pipette  of  drawn-out  glass  tubing  similar  to  that  illus- 
trated in  Fig.  n,  except  that  the  drawn-out  part  is  wider 
and  shorter.  Three  rows  of  7  x  75  mm.  test  tubes  are  then 
set  up  and  further  dilutions  made  according  to  the  follow- 
ing scheme; 

Water  Serum         Bacterial  suspension    Dilution  equals 

.o  drop  10  drops  15  drops  1-25 

5  drops  5  drops  15  drops  1-50 

8  drops  2  drops  15  drops  1-125 

9  drops               i  drop  15  drops  1-250 
10  drops  o  drop  15  drops  control 


AGGLUTININS  AND  PRECIPITINS 


97 


The  three  rows  of  tubes  are  set  up  so  as  to  use  suspensions  in  each  row 
of  bacillus  typhosus,  paratyphosus  A,  and  paratyphosus  B.  The  dilutions 
may  be  carried  further  if  necessary.  The  tubes  are  incubated  in  a  water  bath 
at  55°  C.  for  two  hours,  are  read  immediately,  and,  if  desired,  again  after 
twenty-four  hours  in  the  refrigerator.  The  standard  method  of  Dreyer  may 
be  adapted  to  other  methods  of  dilution  and  incubation,  but  must  be  the 
same  in  the  study  of  every  case. 

In  unvaccinated  individuals  agglutination  in  a  dilution  of  1-25 
against  bacillus  typhosus  justifies  suspicion,  and  if  marked  in  dilu- 
tion of  1—50  is  nearly  always  diagnostic.  Browning  offers  the  fol- 
lowing table  as  indicating  positive  reactions  in  each  of  the 
diseases  indicated. 


Organism 

B.  typhosus 

B.  paratyphosus  A. 

B.  paratyphosus  B. 


Serum  dilution 
I-IOO 

1-50  (or  even  lower  1-20) 
1-200 


These  criteria  are  not  applicable  to  vaccinated  persons  or  those 
who  have  previously  had  typhoid  or  paratyphoid  fever.  Martin  and 
Upjohn  examined  seventy-five  persons  from;  seven  to  fourteen 
months  after  typhoid  vaccination  and  found  that  the  serum  of  two- 
thirds  agglutinated  bacillus  typhosus  in  serum  dilutions  of  1-200, 
and  that  of  one-tenth  agglutinated  in  dilutions  of  1-800.  These  are 
higher  levels  than  are  usually  reached  by  unvaccinated  persons  dur- 
ing the  course  of  the  disease.  Vaccination  with  typhoid  vaccine 
produces  minor  agglutinins  for  para  A  and  B,  but  in  very  low  con- 
centration. Triple  vaccines  produce  agglutinins  for  para  A  and  B, 
but  rarely  in  dilutions  exceeding  1-50  or  i-ioo.  The  following 
chart,  taken  from  Mackie  and  Wiltshire,  as  quoted  by  Browning, 
illustrates  the  change  in  titer  of  blood  serum  in  the  course  of  infec- 
tion with  bacillus  paratyphosus  A. 


Serum  dilutions 

1—50 

I—  100 

1—200 

I—SOO 

I—  1000 

1—2000 

Fourth  or  fifth  day  of  illness: 
B.  typhosus  

+ 
+  +  + 

+  +  + 
+  +  +  + 
+  + 

+ 

+  + 
+  +  +  + 

+ 
+  +  +  + 

+  +  +  + 

+++ 

+  + 

B.  paratyphosus  A  

B.  paratyphosus  B  
Thirteenth  day  of  illness: 
B.  typhosus       .          .... 

B.  paratyphosus  A  
B.  paratyphosus  B  

The  first  test  in  this  patient  was  strongly  suggestive,  since  it  is 
rare  in  a  vaccinated  individual  to  find  the  titer  for  either  para  A  or  B 
to  exceed  that  of  typhosus.  In  our  experience  typhoid  in  the  vac- 
cinated is  likely  to  show  titers  in  the  first  week  of  1-500  for  typhosus, 
i-ioo  for  para  A,  and  1-50  for  para  B ;  toward  the  end  of  the 
second  week  they  are  likely  to  be,  respectively,  1-2500,  1-750, 
1-250;  and  in  the  third  week  1-3000  or  higher  for  typhosus  with 
slight  increases  for  para  A  and  B.  The  titers  then  subside.  It  will 
be  noted  that  infection  increases  not  only  the  major  but  also  the 
minor  agglutinins. 
7 


98  THE  PRINCIPLES  OF  IMMUNOLOGY 

Space  does  not  permit  a  complete  discussion  of  the  results  of  the 
test,  but  it  may  be  said  that  a  positive  Dreyer  test  indicates  the 
presence  of  some  form  of  enteric  fever.  If,  however,  the  isolation 
of  organisms  from  the  stools  indicates  the  nature  of  the  disease  the 
test  may  sometimes  mislead.  For  example,  we  have  found  para- 
typhosus  B  in  the  stools  of  a  patient  whose  serum  titer  curve  indi- 
cated the  presence  of  a  para  A  infection.  The  test  should  go  hand 
in  hand  with  careful  clinical  study  and  bacteriological  examination 
of  the  blood,  feces,  and  urine. 

Hemagglutinins. — The  agglutination  of  blood-cells  and  other 
body  cells  follows  the  same  general  principles  laid  down  for  bacterial 
agglutinins.  In  the  case  of  agglutinins  for  red  blood-corpuscles  the 
name  hemagglutinins  has  been  adopted.  These  may  be  divided  into 
auto-hemagglutinins,  iso-hemagglutinins,  and  hetero-hemagglu- 
tinins.  The  auto-hemagglutinin  is  contained  in  the  same  blood  as 
the  cells  it  agglutinates,  but  certain  factors  operate  to  prevent  agglu- 
tination in  the  living  body.  For  example,  Rous  and  Robertson  have 
shown  the  presence  in  rabbits,  which  had  received  repeated  small 
blood  transfusions,  of  an  auto-hemagglutinin  which  operates  at 
temperatures  lower  than  that  of  the  animal,  but  on  raising  the  tem- 
perature to  38°  to  40°  C.  the  clumps  break  up  and  a  homogeneous  emul- 
sion results.  The  same  workers  also  demonstrated  the  presence  of 
auto-agglutinins  in  rabbits  subjected  to  repeated  withdrawal  of 
small  quantities  of  blood.  It  has  been  stated  that  this  phenomenon 
may  also  occur  in  acquired  hemolytic  jaundice  (Hayem-Widal 
type),  pernicious  anemia,  malaria,  and  other  diseases,  but  more 
recent  studies  tend  to  contradict  this  statement.  Hornby  states 
that  auto-hemagglutinins  have  been  demonstrated  frequently  in 
animals  infected  with  trypanosomes.  Hetero-agglutinins  were  dis- 
covered by  Creite  and  Landois,  who  noted  that  the  serum  of  certain 
animals  produced  agglutination  when  brought  in  contact  with  the 
cells  of  certain  other  species;  for  example,  the  serum  of  the  goat 
and  the  erythrocytes  of  rabbit,  man,  or  pigeon.  Bordet  discovered 
in  the  course  of  his  studies  on  hemolysins  that  if  an  animal  is  im- 
munized with  the  erythrocytes  of  another  species,  the  blood  serum 
will  contain  not  only  hemolysin,  but  also  hemagglutinin  for  the 
cells  used  in  immunization.  Thus  we  have  to  consider  normal 
hetero-hemagglutinins  and  immune  hetero-hemagglutinins.  Such 
normal  antibodies  are  present  in  low  titer,  but  immune  agglutinins 
of  this  sort  may  be  induced  up  to  titers  of  several  thousand.  The 
methods  employed  for  the  production  of  such  agglutinins  are  the 
same  as  those  for  producing  hemolysis  and  will  be  considered  under 
that  subject.  The  determination  of  the  titer  of  hemagglutinative 
sera  is  by  essentially  the  same  methods  as  for  bacterial  agglutinins, 
save  that  the  cells  are  washed  as  for  experiments  in  hemolysis,,  and 
usually  a  fixed  percentage  emulsion  of  cells  is  employed.  The  influ- 
ence of  heat  and  other  physical  agents,  as  well  as  chemicals,  is  much 
the  same  as  for  hemolysins  (see  page  115). 


AGGLUTININS  AND  PRECIPITINS 


99 


Iso-hemagglutinms,  Classification. — Iso-hemagglutinins  are 
those  which  exist  in  certain  members  of  a  species  for  cells  of  cer- 
tain other  members  of  the  same  species.  Although  iso-hemagglu- 
tinins  may  somewhat  rarely  occur  in  lower  animals,  they  appear 
with  great  regularity  in  human  blood.  They  were  discovered  in 
1906  by  Landsteiner  and  Shattock,  working  independently. 
Landsteiner,  by  a  study  of  the  interaction  of  sera  and  corpuscles, 
classified  all  human  bloods  in  three  groups  and  determined  that  the 
property  of  iso-agglutinination  is  normal  to  man  and  does  not  vary 
under  pathological  conditions.  Hektoen  noted  in  1907  that  the  three 
groups  do  not  include  all  individuals,  and  in  the  same  year  Jansky 
published  the  classification  in  four  groups.  This  was  confirmed  by 
Hektoen  and  subsequently  adopted  by  Ottenberg.  Moss,  in  1910, 
without  knowledge  of  Jansky's  work,  also  found  that  it  is  necessary 
to  divide  bloods  into  four  groups  in  order  to  include  all  individuals, 
but  unfortunately  employed  a  system  of  numbering  the  groups  the 
opposite  of  that  of  Jansky.  Because  of  the  priority  of  Jansky's  sys- 
tem and  its  important  support  by  Hektoen  and  by  Ottenberg  and 
others,  we  prefer  to  use  it  rather  than  that  of  Moss.  Groups  I  and 
IV  are  transposed  in  the  two  systems  but  Groups  II  and  III  re- 
main the  same,  hence,  groups  are  transposable  from  one  basis  to  the 
other.  The  groups  are  not  present  at  birth,  but  become  established  at 
about  the  end  of  the  first  year  of  life  and  remain  constant  thereafter ;  they 
are  heritable  according  to  the  Mendelian  law.  Disease  does  not  change 
the  group  of  an  individual,  although,  according  to  some  of  our  experi- 
ments, it  seems  possible  that  the  agglutinin  titer  may  be  somewhat 
reduced  by  prolonged  disease.  Jansky  included  in  Group  I  those 
bloods  whose  sera  agglutinate  cells  of  all^^ier  groups  and  whose 
cells  are  not  agglutinated  by  any  sera;  C^l^tV  is  the  reciprocal 
of  Group  I  in  that  the  sera  agglutinate  «  K,  but  the  cells  are 
agglutinated  by  sera  of  all  the  other  groups^^ffoups  II  and  III  are 
reciprocals  of  each  other  and  occupy  intermediate  positions  between 
Groups  I  and  IV.  This  may  be  rendered  clearer  by  the  following  table : 

Group       I.     Serum  agglutinates  cells  II,  III  and  IV. 

Cells  agglutinated  by  no  sera. 
Group     II.     Serum  agglutinates  cells  III  and  IV. 

Cells  agglutinated  by  sera  I  and  III. 
Group  III.     Serum  agglutinates  cells  II  and  IV. 

Cells  agglutinated  by  sera  I  and  II. 
Group   IV.     Serum  agglutinates  no  cells. 

Cells  agglutinated  by  sera  I,  II  and  III. 

The  following  chart  presents  the  classification  graphically;  the 
+  sign  indicates  agglutination : 

JANSKY  CLASSIFICATION 

Sera 

I.                II.               III.              IV. 
j  

5          II.  +  —  +  — 

3      in.  +  - 

IV.  -f  - 


100 


THE  PRINCIPLES  OF  IMMUNOLOGY 


Inasmuch  as  the  Moss  classification  has  been  widely  adopted  we 
include  the  chart  of  that  system  so  as  to  show  the  relation  of  the 
two  systems  of  grouping  : 

Moss  CLASSIFICATION 


Sera 
ii. 


I. 

II. 

III. 

IV. 


III. 


IV. 

+ 
+ 


It  is  of  the  utmost  importance  that  when  the  groups  are  deter- 
mined in  any  individual  the  method  of  classification  should  be 
clearly  stated. 

The  incidence  of  the  groups  varies  somewhat,  according  to  the 
figures  of  different  investigators,  and  there  is  probably  a  factor  of 
error  due  to  "  random  sampling,"  in  spite  of  the  large  number  of 
individuals  examined.  Selected  figures  follow,  according  to  the 


Jansky  classification : 


Groups 


II. 


III. 


IV. 


per  cent. 

47- 

per  cent. 

ii. 

per  cent. 

6. 

per  cent. 

per  cent. 

40. 

per  cent. 

7- 

per  cent. 

10. 

per  cent. 

per  cent. 

39- 

per  cent. 

13. 

per  cent. 

2. 

per  cent. 

per  cent. 

42.4 

per  cent. 

8.3 

per  cent. 

3-i 

per  cent. 

per  cent. 

38.5 

per  cent. 

12.5 

per  cent. 

6. 

per  cent. 

I. 

Von  Dungern 
Hirschfeld    .   36. 

Moss    43. 

Olmstead  46. 

Karsner  46.2 

Koeckert  43. 

Average    42.84  per  cent.      41.38  per  cent.     10.36  per  cent.     5.42  per  cent. 

The  table  shows  that  about  four-fifths  of  all  individuals  fall  in 
Groups  I  and  II,  about  equally  divided  between  the  two  groups,  the 
next  most  frequent  being  Group  III,  and  the  least  frequent  being 

Characters  of  /•  magglutinins. — The  iso-hemagglutinins  are 
neither  filterable  noi^B^zable,  and  are  destroyed  by  heat  of  62°  to 
66°  C.  for  thirty  minutes,  depending  on  concentration,  i.e.,  the  agglu- 
tinins  in  high  dilutions  (1-32,  1-64)  disappear  at  62°  C.,  and  in  the 
undiluted  sera  at  65°  to  66°  C.  They  are  present  in  transudates  and 
exudates  as  well  as  in  the  plasma  and  serum,  the  serum  showing  a 
greater  concentration  than  the  plasma.  In  serum  the  titer  is  usu- 
ally between  1-16  and  1-32,  although  it  may  be  as  low  at  1-2,  and 
has  been  reported  as  high  as  1-320,  irrespective  of  group.  There  is 
variation  of  agglutinin  content  and  probably  of  agglutinability  of 
cells  at  different  times  in  the  same  individual. 

The  fact  that  a  blood  contains  an  iso-agglutinin  does  not  nec- 
essarily mean  that  it  will  similarly  dissolve  corpuscles,  but  the 
converse  is  true ;  namely,  that  if  a  serum  shows  iso-hemolytic  prop- 
erties it  is  also  iso-hemagglutinative ;  the  group  relationship  prevails 
in  both  agglutination  and  hemolysis.  In  fact,  agglutination  always 
precedes  hemolysis.  In  spite  of  this  generally  accepted  view, 
Kolmer  claims  recently  to  have  demonstrated  the  presence  of  iso- 
hemolysins  independent  of  iso-agglutinins. 


AGGLUTININS  AND  PRECIPITOUS  A  L  \\ 

The  Mechanism  of  Iso-hemagglutination. — Numerous  theories 
have  been  offered,  of  which  we  present  that  of  Landsteiner.  It  has 
recently  received  support  in  this  laboratory  by  the  painstaking  spe- 
cific absorption  experiments  of  Koeckert.  Landsteiner  considers 
that  the  division  into  four  groups  depends  upon  the  presence,  dif- 
ferently distributed  in  bloods,  of  two  agglutinins,  a  and  b,  and  two 
agglutinogens,  A  and  B.  The  distribution  of  these  may  be  tabulated 
as  follows  (Jansky  classification)  : 

Group  Agglutinins  (serum)      Agglutinogens  (cells) 

I.  a    b 

II.  —   b  A  — 

III.  a   —  —  B 

IV.  A  B 

Aside  from  the  support  offered  by  Koeckert,  in  his  demonstration 
that  specific  absorption  experiments  prove  the  presence  of  these 
bodies,  further  confirmatory  evidence  is  found  in  the  fact  that  the 
agglutinogenic  character  of  cells  is  demonstrable  in  the  early  months 
of  life,  whereas  the  agglutinins  do  not  appear  until  near  the  end  of 
the  first  year.  It  is  also  stated  that  transfusion  with  a  certain  group 
may  lead  to  the  development  in  the  recipient  of  specific  iso-agglu- 
tinins  for  the  group  injected.  Kolmer's  work,  however,  shows  that 
immunization  of  animals  with  the  blood  of  the  various  groups  pro- 
duces a  hemagglutinative  and  hemolytic  serum  without  group  char- 
acters. Karsner  and  Koeckert  have  shown  that  desiccation  leads  to 
a  loss  of  specificity  of  the  sera,  and  that  at  a  certain  period  in  the 
desiccation  a  common  agglutinin  is  found  which  clumps  the  cells  of 
all  groups,  including  Group  I.  This  is  probably  in  part  due  to 
alterations  in  physical  character  of  the  redissolved  sera,  and  to 
alterations  in  hydrogen  ion  concentration,  as  shown  by  Karsner 
and  Collins.  Therefore,  although  the  Landsteiner  hypothesis  offers 
an  excellent  working  basis,  it  seems  probable  that  an  intricate 
physico-chemical  mechanism  is  largely  concerned  in  the  phenom- 
enon of  iso-hemagglutination. 

Iso-hemagglutinins  in  Lower  Animals. — The  presence  of  iso- 
hemagglutinins  in  animals  other  than  man  is  extremely  irregular 
and  infrequent.  Certainly  no  classification  into  definite  groups  has 
so  far  been  demonstrated.  In  our  own  experience  the  examination 
of  from  ten  to  twenty  members  each  of  dog,  rabbit,  cat,  and  guinea- 
pig  species  has  failed  to  show  iso-hemagglutinins,  but  others  who 
have  examined  larger  numbers  have  found  an  occasional  instance 
of  iso-hemagglutination. 

Relation  of  Iso-hemagglutinins  to  Blood  Transfusion. — The 
principal  importance  of  iso-hemagglutinins  and  the  related  iso-hemo- 
lysins  in  human  medicine  relates  to  the  transfusion  of  blood,  a  thera- 
peutic measure  which  civil  and  military  practice  have  shown  to  be 
of  the  utmost  value  in  combating  secondary  anemia  following 
hemorrhage.  It  is  also  recommended  for  prolonged  sepsis  with  or 
without  severe  anemia,  for  primary  anemias,  and  for  certain  other 


PRINCIPLES  OF  IMMUNOLOGY 


diseases,  but  results  are  not  so  brilliantly  successful  as  in  secondary 
anemias,  particularly  those  resulting  from  acute  hemorrhage.  Ill 
effects  following  transfusion  are  spoken  of  as  reactions  and  include 
fever,  chills,  cyanosis,  hemoglobinuria,  and  even  death.  Cases  com- 
ing to  autopsy  show  parenchymatous  degenerations  of  solid  organs, 
marked  congestion  of  all  viscera,  acute  splenic  hyperplasia,  hemo- 
globin staining,  and  sometimes  multiple  small  emboli  of  agglutin- 
ated erythrocytes.  Blood  studied  in  life  has  shown  phagocytosis  of 
erythrocytes  by  the  recipient's  white  corpuscles.  The  reactions  de- 
pend in  large  part  on  intravascular  agglutination  and  hemolysis, 
but  probably  certain  other  factors  play  a  part.  The  prevention  of 
these  other  factors  awaits  the  determination  of  their  nature,  but  the 
avoidance  of  agglutination  and  hemolysis  can  easily  be  accomplished 
by  use  of  the  very  simple  tests  for  the  determination  of  the  presence 
of  conflicting  iso-agglutinins.  The  simplest  of  these  tests  is  the  deter- 
mination of  the  groups  to  which  recipient  and  prospective  donors 
belong.  The  most  desirable  means  of  selection,  in  our  opinion,  is 
that  whereby  the  donor  is  chosen  from  the  same  group  as  the  patient. 
Lee  and  others  have  maintained  that  it  is  equally  safe  to  use  members 
of  Group  I  as  donors  for  recipients  of  any  group.  The  argument  in 
favor  of  this  procedure  is  based  on  the  statement  that  the  real  danger 
in  transfusion  is  the  use  of  a  donor  whose  cells  are  agglutinated  by  the 
recipient's  plasma  and  that  the  converse  has  little  or  no  significance. 
The  cells  of  Group  I  are  not  agglutinated  by  any  sera  and  are,  there- 
fore, safe  to  use.  In  our  own  experience  we  have  seen  occasional 
reactions  following  this  procedure  and  prefer  to  use  a  donor  in  the  same 
group  as  the  recipient.  Reactions  following  the  general  use  of  Group  I 
donors  do  not  necessarily  mean  that  the  trouble  is  the  result  of  agglu- 
tination or  hemolysis,  for,  as  has  been  indicated  above,  other  factors 
may  be  concerned.  Nevertheless,  it  holds  true  that  thousands  of  trans- 
fusions have  been  done  with  Group  I  as  the  "  universal  "  donor  and 
without  reaction.  The  explanation  of  the  fact  that  a  donor  may  thus 
be  used,  whose  plasma  or  serum  is  capable  of  agglutinating  the  recipi- 
ent's erythrocytes  in  vitro,  is  not  settled,  but  certain  theories  have 
been  offered.  It  must  be  remembered  that  in  transfusion  a  small  bulk 
of  blood  is  introduced,  as  compared  with  the  total  bulk  in  the  recipi- 
ent's body.  Therefore,  agglutinins  introduced  in  this  way  are  much 
diluted,  and  as  they  ordinarily  occur  in  low  titer  they  may  be  sufficiently 
diluted  to  be  ineffective.  Another  possibility  is  that  the  agglutinins 
are  absorbed  equally  by  an  extremely  large  number  of  cells,  each  cell, 
therefore,  taking  up  too  small  an  amount  to  be  subjected  to  agglutina- 
tion. A  third  possibility  is  that  an  excess  of  non-agglutinable  cells 
and  the  presence  of  the  patient's  own  plasma  permits  of  the  formation 
of  only  small  clumps  of  cells,  so  small  that  they  are  of  no  significance 
in  the  circulation.  Our  own  work  has  failed  to  demonstrate  anti- 
agglutinins  in  a  large  number  of  tests,  and  it  seems  improbable  that  a 
mechanism  of  this  type  operates  to  protect  the  recipient.  It  is  conceiv- 
able, however,  that  these  possible  factors  of  safety  may  not  operate  and 


AGGLUTININS  AND  PRECIPITINS  103 

reaction  follow  this  type  of  transfusion.     We  cannot  enter  here  into 
a  discussion  of  methods  of  transfusion. 

Methods  for  Testing  Human  Blood. — The  simplest  method  depends  upon 
the  preservation  in  the  laboratory  of  known  Group  II  and  Group  III  sera.  These 
should  be  selected  so  that  they  have  a  relatively  high  titer,  and  should  not  be 
employed  if  they  titrate  less  than  i  to  16.  The  method  to  be  described  is  essen- 
tially that  of  Lee  and  Minot.  The  apparatus  includes  a  few  7x75  mm.  test-tubes, 
a  platinum  loop,  microscope  slides  with  at  least  one  built  up  on  the  ends  with 
pieces  of  glass  rod  or  match  sticks  glued  on  by  means  of  balsam  so  that  another 
slide  may  be  inverted  upon  it  with  hanging  drops.  A  microscope  is  useful  but 
not  essential,  since  a  hand  lens  of  10  diameters  magnification  is  satisfactory.  A 
small  moist  chamber  is  desirable  but  not  essential.  In  well  equipped  labora- 
tories the  serum  may  be  kept  in  the  ice  chest  in  sterile  ampoules  or  small  bottles 
and  drops  removed  as  required.  Somewhat  more  satisfactory  is  preservation 
in  sections  of  drawn  out  glass  tube  similar  to  that  used  for  vaccine  virus.  Each 
small  tube  contains  serum  for  one  test  and  the  serum  may  be  blown  out  exactly 
as  is  done  with  vaccine  virus.  Phenol  0.5  per  cent,  may  be  used  as  a  pre- 
servative. One-half  cubic  centimeter  of  physiological  salt  solution  is  placed  in  a 
test-tube,  and  to  this  are  added  one  or  two  drops  of  blood,  obtained  by  ear  or 
ringer  puncture,  sufficient  to  make  a  slightly  opaque  emulsion.  Clotting  of  the 
mixture  is  not  harmful  since  subsequent  shaking  of  the  tube  will  produce  a 
homogeneous  suspension.  Upon  a  microscope  slide  are  placed  one  drop  eacli  of 
the  sera  of  Groups  II  and  III.  With  the  platinum  loop  a  drop  of  blood  suspension 
is  mixed,  by  gentle  rubbing,  in  each  of  the  serum  drops  and  the  slide  immedi- 
ately inverted  upon  the  prepared  slide  or  a  small  rack  so  as  to  make  hanging 
drops.  At  the  end  of  five  or  ten  minutes  the  reaction  occurs  and  may  be  seen 
with  the  naked  eye ;  in  order  to  avoid  mistakes  owing  to  slight  agglutination  it 
is  important  to  observe  with  the  16  mm.  lens  of  the  microscope  or  a  hand 
lens.  If  a  small  number  of  specimens  is  examined  it  is  well  to  have  controls 
with  known  I,  II  or  III  cells.  If  the  reaction  is  delayed  the  slide  should  be  kept 
in  a  moist  chamber  for  one-half  hour  and  then  observed.  The  group  to  which 
the  cells  belong  is  determined  by  the  following  section  from  the  chart  of 
inter-agglutination : 

STANDARD  SERA 

II.  III. 

2  I.  _ 

3  n-  -  + 

CJ     III.  +  - 

IV.  +  + 

Thus  if  the  cells  are  agglutinated  by  both  sera  they  belong  to  Group  IV;  if 
not  agglutinated  at  all  and  the  control  cells  show  that  the  sera  agglutinate  prop- 
erly the  cells  belong  to  Group  I;  if  agglutinated  by  only  III  serum  they  belong  to 
Group  II,  and  if  agglutinated  by  only  II  serum  they  belong  to  Group  III. 

Hanging  drops  are  not  essential,  but  serve  to  make  the  reaction  somewhat 
clearer.  The  reaction  occurs  with  the  slides  upright.  In  this  case  cover  slips 
may  be  used.  Many  employ  undiluted  blood  and  cover  with  cover  slips,  but 
rouleaux  formation  sometimes  offers  a  confusing  picture. 

It  has  been  suggested  that  since  the  important  point  of  determination  is  as 
to  whether  or  not  the  donor's  corpuscles  are  agglutinated  by  the  patient's  serum, 
the  latter  may  be  separated  and  placed  on  a  slide  with  the  donor's  corpuscles. 
The  separation  of  the  serum  requires  more  time  than  a  complete  test  as  given 
above,  and  is  subject  to  serious  error  if  the  patient's  serum  happens  to  be  of  low 
agglutinin  titer. 

If  standard  sera  are  not  available  they  may  be  prepared  if  a  known  II  or 
III  blood  can  be  obtained.  The  interaction  of  the  cells  and  serum  with  fifteen  or 
twenty  other  bloods  can  be  worked  out  on  the  basis  of  the  chart  on  inter- 
agglutination.  Space  does  not  permit  of  giving  the  details,  but  Brem's  method 
gives  them  accurately.  If  this  cannot  be  done  the  method  of  Rous  and  Turner 
is  probably  the  best  of  the  methods  for  use  where  standard  sera  are  not  to  be 
had,  since  this  method  determines  the  activity  of  both  the  cells  and  serum  of 
the  donor  and  recipient.  The  method  with  slight  omissions  is  taken  directly 
from  the  article  of  Rous  and  Turner  in  volume  64  of  the  Journal  of  the  Amer- 
ican Medical  Association. 


104  THE  PRINCIPLES  OF  IMMUNOLOGY 

"  Collection  of  the  Blood. — The  blood  is  taken  from  the  patient  and  the  pros- 
pective donors  in  a  I— 10  mixing  pipette,  such  as  is  used  in  counting  leucocytes. 
The  pipette  is  rinsed  beforehand  with  10  per  cent,  sodium  citrate  in  water;  the 
citrate  solution  is  drawn  up  to  the  mark  i ;  the  pipette  is  rapidly  filled  with  blood 
from  a  puncture  of  the  ear  or  finger ;  and  without  pause  the  mixture  is  expelled 
into  a  small,  narrow  test-tube.  There  is  thus  obtained  a  citrated  blood  containing 
slightly  less  than  I  per  cent,  of  citrate.  The  pipettes  which  we  have  employed  hold 
only  0.25  c.c.  of  fluid.  This  much  blood  is  easily  obtained  from  a  single  puncture. 
There  is  no  objection  to  increasing  the  flow"  by  pressure.  Should  it  cease  before 
the  pipette  is  full,  the  blood  must  be  at  once  expelled  into  a  test-tube,  in  order 
that  it  may  mix  with  the  citrate  and  clotting  be  avoided.  The  mixture  is  then 
taken  up  again,  a  new  puncture  made,  and  the  pipette  completely  filled.  After 
each  blood  is  obtained,  the  pipette  is  rinsed  with  citrate,  then  with  distilled  water, 
then  with  fresh  citrate,  and  it  is  ready  for  another  blood.  If  several  donors  are 
to  be  tested,  two  pipettefuls  of  Citrated  blood  should  be  obtained  from  the  patient. 
It  is  best  to  take  them  from  different  puncture  wounds,  in  order  to  avoid  a.  pos- 
sible clotting  in  the  pipette. 

"  Mixing. — The  mixing  is  done  in  pipettes  with  a  capillary  end — the  so-called 
Wright  pipettes  obtained  by  drawing  out  glass  tubing  in  the  flame.  (Fig.  u.) 
The  citrated  bloods  are  used  as  such,  and  two  combinations  are  made  of  the 
patient's  blood  with  that  of  each  prospective  donor,  a  mixture  containing  nine 
parts  of  the  patient's  blood  to  one  of  the  donor's,  and  a  mixture  of  equal  parts 
of  the  two.  The  proportions  used  need  be  only  approximate.  In  case  of 
emergency  the  first  of  the  mixtures  will  suffice,  since  by  its  use  the  most 
dangerous  possibility,  namely,  that  the  blood  of  the  recipient  might  destroy  that 
of  the  donor,  can  be  ruled  out.  Following  the  technic  usual  with  Wright  pipettes, 
the  capillary  tube  is  marked,  blood  is  drawn  to  the  mark,  and  each  column  of 
the  blood  is  separated  by  an  air  bubble  from  the  next  that  is  drawn  up.  To 
insure  proper  mingling,  each  mixture  should  be  expelled  on  a  slide,  or  Widal 
plate,  and  then  drawn  high  in  the  pipette,  which  may  be  sealed  off  in  the  flame 
in  case  the  examination  is  not  to  be  made  for  some  time. 

"  Incubation. — No  incubation  in  the  ordinary  sense  is  necessary.  The  pipettes 
are  kept  at  room  temperature,  and  readings  are  begun  after  two  minutes  if 
there  is  need  to  hurry.  Readings  are  for  agglutination,  and  even  within  two 
minutes  this  is  plainly  evident,  except  when  the  agglutinating  forces  are  notably 
weak.  In  the  final  choice  of  a  donor  it  is  safest  to  rely  on  results  obtained  after 
the  mixtures  have  stood  for  fifteen  minutes.  But  the  ruling  out  of  individuals 
with  unfit  blood  may  be  begun  practically  at  once. 

"  Readings. — The  capillary  end  of  each  pipette  is  broken,  a  small  drop  of  the 
blood  expressed  on  a  slide,  a  large  drop  of  normal  salt  solution  superimposed 
without  mixing,  a  coverslip  put  on,  and  the  preparation  examined  for  agglutin- 
ation under  the  microscope.  Fresh  preparations  can  be  made  at  intervals  if  de- 
sired. The  salt  solution  is  not  absolutely  necessary ;  but  very  clear  pictures  are 
obtained  as  the  blood  spreads  in  it.  When  agglutination  has  occurred,  the  red 
cells  show  a  characteristic  clumping,  sometimes  in  small  masses,  often  in  large 
ones  that  are  very  evident  microscopically. 

"  If  there  is  no  clumping  in  the  preparations  made  after  the  mixtures  have 
stood  fifteen  minutes,  the  assumption  is  warranted  that  the  bloods  do  not  agglu- 
tinate or  hemolyze  each  other.  But  if  clumping  is  present  in  the  9-1  mixture 
and  to  a  less  degree  or  not  at  all  in  the  i-i  mixture,  it  is  certain  that  the  blood 
of  the  patient  agglutinates  that  of  the  donor,  and  may  perhaps  hemolyze  ^it. 
Transfusions  in  such  cases  are  dangerous.  Clumping  in  the  i-i  mixture  with 
little  or  none  in  the  9-1  indicates  that  the  plasma  of  the  prospective  donor 
agglutinates  the  cells  of  the  prospective  recipient.  For  practical  purposes  these 
findings  suffice.  But  if  there  is  a  desire  to  know  whether  both  bloods  contain 
agglutinins,  a  1-9  mixture  should  be  made.  If  this  and  the  9-1  mixture  show 
large  clumps,  whereas  the  clumps  are  smaller  when  the  bloods  are  mixed  in  equal 
parts,  two  agglutinins  must  be  present.  Should  there  be  only  one  agglutinin, 
little  clumping  or  none  will  be  observed  when  the  blood  containing  the  agglutinin 
is  diluted  with  nine  parts  of  the  other  blood." 

By  the  use  of  the  technic  indicated  in  the  last  paragraph,  it  is 
possible  to  overcome  error  due  to  weak  agglutinin  content  of  the  re- 
cipient's blood.  This  we  believe  is  of  especial  importance  if  the 
patient  has  been  ill  for  a  long  time. 


AGGLUTININS  AND  PRECIPITINS  105 

Reactions  to  Transfusion. — The  effects  on  the  body  of  introducing 
high  titer  hemagglutinins  have  been  studied  experimentally  in  normal 
animals  and  in  those  which  have  been  splenectomized.  In  normal  animals 
there  is  found  agglutination  of  red  blood-corpuscles  with  embolism  in 
liver,  lungs  and  other  viscera.  The  liver  is  often  found  to  show  hyaline 
necrosis  in  connection  with  the  emboli.  The  spleen  is  large  and  dif- 
fluent, and  there  are  small  areas  of  necrosis  as  well  as  phagocytosis  of 
erythrocytes  by  endothelial  cells.  Necrosis  is  also  found  in  the  fol- 
licles of  lymph-nodes.  Multiple  hemorrhages  may  also  be  noted.  After 
splenectomy  the  phagocytic  function  of  the  spleen  is  taken  over  by  the 
lymph-nodes.  The  incident  hemolysis  in  either  case  leads  to  hemo- 
globinemia  and  hemoglobinuria,  but  in  splenectomized  animals  the 
threshold  of  excretion  of  hemoglobin  is  somewhat  higher  than  in 
normal  animals. 

In  severe  and  fatal  reactions  in  man  the  phenomena  are  not  likely 
to  be  so  marked.  Phagocytosis  of  erythrocytes  by  the  recipient's  leuco- 
cytes has  been  observed.  We  have  performed  autopsies  on  twelve 
cases  in  which  transfusion  was  practised  shortly  before  death,  in  three 
of  which  the  death  was  at  least  in  part  due  to  the  use  of  unsuitable 
blood.  In  all  of  these  the  spleen  was  considerably  enlarged  (170, 
390,  400  grams),  and  in  one  there  were  multiple  small  hemorrhagic 
infarcts.  One  case  showed  enlarged  soft  white  lymph-nodes.  The 
bone  marrow  was  normal  in  all.  All  showed  marked  cloudy  swelling 
of  the  kidneys.  Two  showed  hemoglobinuria,  and  in  one  of  these 
there  was  post-mortem  staining  by  hemoglobin.  In  the  case  with 
multiple  infarcts  of  the  spleen  50  c.c.  Group  II  blood  had  been  given 
to  a  Group  III  recipient,  and  there  was  neither  hemoglobinemia  nor 
hemoglobinuria.  In  the  case  with  hemoglobinemia  and  hemoglobin- 
uria about  700  c.c.  Group  III  blood  was  given  a  Group  I  recipient. 
Unfortunate  accidents  led  to  these  errors,  and  the  groups  were  dis- 
covered subsequent  to  the  operations.  In  the  nine  cases  where  the 
transfusions  were  satisfactory  the  spleen  was  either  normal  or  if 
enlarged  was  accompanied  by  septicemia. 

Chemical  Agglutination  of  Erythrocytes. — Blood-corpuscles  are 
agglutinated  not  only  by  various  sera  but  also  by  certain  chemical  sub- 
stances. Gay  has  examined  the  function  of  the  tonicity  of  the  sur- 
rounding medium  in  determining  iso-hemagglutination  and  maintains 
that  the  bloods  of  that  group  whose  cells  are  non-agglutinable  (Group 
I)  are  constantly  of  higher  total  molecular  concentration  than  the 
other  bloods.  He  further  states  that  a  "  simple  hypertonic  solution 
of  CaCl2,  but  more  particularly  solutions  hypertonic  both  in  respect  to 
NaCl  and  CaCl2,  produces  a  cohesion  of  any  human  blood  after  sev- 
eral hours  resembling  iso-agglutination."  Studies  of  hypotonic  solu- 
tions and  of  variations ,  of  any  considerable  degree  in  hydrogen  ion 
concentration  have  been  rendered  difficult  because  hemolysis  is  likely 
to  occur  under  these  conditions  and  render  conclusions  difficult.  Land- 
steiner  and  Jagic  in  1904  were  the  first  to  call  attention  to  the  fact  that 
a  well-defined  colloid,  namely  silicic  acid,  agglutinates  erythrocytes. 


io6  THE  PRINCIPLES  OF  IMMUNOLOGY 

Gengou  reported  agglutination  and  hemo lysis  by  means  of  such  chemi- 
cal precipitates  as  calcium  fluoride  and  barium  sulphate,  but  in  these 
instances  serum  served  to  prevent  agglutination.  This  appears  to  be 
another  example  of  protective  colloidal  action.  According  to  Girard, 
Mangin  and  Henri,  the  red  cells  carry  electro-negative  charges,  but 
agglutination  has  been  produced  by  colloids  regardless  of  the  electrical 
charge  they  carry.  We,  in  collaboration  with  Hanzlik,  have  exam- 
ined a  wide  variety  of  colloids  and  have  determined  that  many  of 
those  which  produce  thrombosis  upon  intravenous  injection  into  animals 
also  produce  agglutination  in  the  test  tube. 

Conglutination. — Bordet  and  Gay,  as  well  as  Muir  and  Browning, 
independently  described  in  1908  the  phenomenon  of  conglutination — an 
agglomeration  of  corpuscles  in  the  presence  of  two  normal  sera.  The 
result  of  this  reaction  is  the  agglutination  of  corpuscles,  but  what  is 
known  of  its  mechanism  makes  it  advisable  to  consider  the  phenomenon 
after  the  discussion  of  hemolysis  (see  page  126). 

PRECIPITATION 

Introduction. — The  discovery  of  agglutination  led  to  the  discovery 
by  R.  Kraus  in  1897  of  the  precipitin  reaction.  His  problem  was  to 
determine  whether  or  not  agglutinating  sera  would  act  in  any  way  on 
extracts  of  bacteria,  and  in  his  work  with  typhoid  bacilli  and  cholera 
vibrios  he  found  that  the  addition  of  the  specific  antisera  to  the  bac- 
terial extracts  led  to  the  formation  of  a  precipitate  and  that  this 
reaction  is  specific.  This  was  confirmed  by  Nicolle.  Previously  Widal, 
Levy  and  Bruns  had  shown  the  converse,  namely,  that  filtrates  of 
typhoid  and  cholera  cultures  upon  injection  led  to  the  formation  of 
agglutinins.  In  1899  Tchistovichs  published  the  results  of  his  work 
with  horse  serum  and  eel  serum,  demonstrating  the  formation  of 
specific  precipitates  when  the  serum  of  rabbits  previously  inoculated 
with  these  sera  was  added  to  the  antigenic  sera.  Bordet  confirmed 
this  with  chicken  serum  and  later  showed  that  cow's  milk  upon  injec- 
tion induces  the  formation  of  a  specific  precipitating  serum  for  the 
casein  of  the  milk.  Kraus  states  that  previous  to  the  publications  of 
Tchistovitchs  and  of  Bordet  he  had  also,  in  collaboration  with  Winter- 
berg  and  E.  P.  Pick,  experimented  with  proteins  of  animal  origin. 
Fish  demonstrated  the  specificity  of  various  milk  antisera  for  their 
respective  antigens.  The  reaction  was  enlarged  in  scope  for  various 
other  animal  proteins.  Kowarski  showed  that  the  reaction  is  specific 
for  higher  vegetable  proteins  as  well  as  for  those  of  bacteria.  Certain 
authors  have  claimed  that  peptones,  globulins,  albumoses  and  other 
protein  products  are  antigenic  in  a  similar  manner,  but  the  weight  of 
evidence  is  that  the  whole  protein  molecule  is  necessary.  A  recent 
review  of  the  literature  on  this  subject  by  Fink  has  shown  that  state- 
ments in  regard  to  the  proportion  of  the  entire  protein  molecule  neces- 
sary to  take  part  in  the  reaction  are  confusing  and  obscure.  Frequently, 
instead  of  testing  against  the  decomposition  product  itself,  the  serum 
obtained  by  its  use  has  been  tested  against  the  entire  protein  molecule. 


AGGLUTININS  AND  PRECIPITINS  107 

Fink  worked  with  the  precipitates  obtained  by  salting  protein  solutions 
and  found  that  rabbits  inoculated  with  one-fourth,  one-third,  one-half, 
and  two-thirds  saturation  products  produced  no  precipitins  nor  com- 
plement-fixing bodies.  In  guinea-pigs,  however,  the  three-fourths 
saturated  and  completely  saturated  products  possess  slight  sensitizing 
and  intoxicating  properties,  the  latter  being  apparently  the  more 
active.  Nevertheless,  three-fourths  saturated  and  completely  saturated 
products  of  egg-white  were  sufficient  to  produce  definite  formation  of 
precipitin  and  complement-binding  antibodies  but  not  in  as  high  a  titer 
as  entire  protein. 

Nature  of  the  Reaction. — In  analogy  with  the  terms  used  in  the 
phenomenon  of  agglutination  Kraus  named  the  antigen,  precipitinogen 
and  the  immune  body  precipitin.  The  reaction  is  similar  to  agglutina- 
tion in  all  respects  save  that  here  we  have  to  deal  with  proteins  in 
solution.  Aging  or  heating  leads  to  the  formation  of  precipitoids, 
group  reactions  as  well  as  inhibition  zones  appear,  heat  has  much  the 
same  influence  in  all  respects  as  in  agglutination,  salts  play  an  im- 
portant part  in  the  reaction  and  specific  absorption  can  be  demonstrated. 
It  is  known,  however,  that  some  protein  molecules  are  largely  built 
up  of  alkaline  amino-acids  and  that  others  are  built  up  largely  of  the 
acid  amino-acids.  Salmine,  for  example,  a  product  of  the  spermatozoa 
of  certain  fish,  consists  almost  entirely  of  strongly  alkaline  amino-acids. 
Gliadine  of  wheat  is  chiefly  built  up  of  dibasic  amino-acids,  glutaminic 
acid.  The  fermentation  end  product  of  salmine  is  alkaline  and  of  gli- 
adine  acid  in  nature.  An  antigliadine  serum  gives  with  a  homologous 
precipitinogen,  a  beautiful  precipitate,  while  a  mixture  of  salmine  and 
antisalmine-serum  gives  no  visible  precipitate.  This  would  indicate 
that  the  alkaline  salts  are  of  importance  in  the  actual  formation  of  the 
precipitins,  and  we  know  by  simple  titration  that  during  the  precipitin 
reaction  there  occurs  a  reduction  of  acidity.  Nevertheless,  it  is  also 
asserted  that  when  the  acidity  is  due  to  an  organic  acid  or  acid  salt 
the  reaction  appears  to  be  promoted.  The  precipitin  is  precipitated  in 
the  euglobulin  fraction  of  the  serum,  is  destroyed  slowly  by  trypsin 
and  rapidly  by  pepsin.  The  immune  serum  contains  the  precipitin 
which  constitutes  the  bulk  of  the  precipitate,  the  latter  thus  represent- 
ing, according  to  Wells,  "  the  insoluble  modification  of  the  previously 
dissolved  precipitin  and  originates  chiefly  or  entirely  in  the  proteins 
of  the  immune  serum."  Welch  and  Chapman  obtained,  with  a  precipi- 
tinogen containing  only  i  gram  of  protein,  a  precipitate  containing  21.1 
grams  of  protein.  Pick  employed  a  precipitinogen  which  did  not  give 
the  biuret  reaction  and  with  this  obtained  a  voluminous  albuminous 
precipitate.  It  must  not  be  understood  that  precipitins  are  always  the 
result  of  immunization,  for  Vaughan  states  that  goat  serum  contains 
a  normal  precipitin  for  rabbit  and  for  dog  sera.  Such  normal  pre- 
cipitins are  not  of  very  high  titer  and  are  not  so  sharply  specific  as 
the  immune  precipitin.  Puppies,  kittens  and  rabbits  up  to  ten  days 
old  may  absorb  native  protein  from  the  milk  of  the  mother  which 
apparently  stimulates  the  formation  of  precipitins.  Sera  of  human 


io8  THE  PRINCIPLES  OF  IMMUNOLOGY 

infants  have  been  observed  to  precipitate  the  protein  of  cow  milk.  It 
appears  possible,  then,  that  from  absorption  through  the  intestinal  tract 
early  in  life  the  protein  may  appear  in  the  circulating  fluid  in  native 
form  and  thus  stimulate  the  formation  of  precipitins.  These,  of 
course,  are  not  normal  precipitins  in  the  sense  indicated  above  for  goat 
serum  but  similarly  are  always  precipitins  of  low  titer  and  not 
highly  specific. 

Experimental  Demonstration. — For  practical  demonstration  of  the  reaction 
the  serum  proteins  are  the  simplest  to  use.  For  immunization  of  animals  the 
intravenous  route  is  by  far  the  best,  injecting  2.0  c.c.  serum  at  five-day  intervals 
and  bleeding  ten  days  after  the  last  dose.  Three  doses  are  usually  sufficient,  but 
five  doses  frequently  give  a  precipitin  of  very  high  titer.  In  order  to  get  clear 
serum  it  is  necessary  to  fast  the  animal  for  twenty-four  hours  before  bleeding, 
thus  eliminating  fat  from  the  serum.  Rabbits  are  the  animals  usually  selected 
for  this  purpose  because  of  their  availability  in  the  laboratory  and  because  of  the 
relative  ease  of  intravenous  injection.  Hektoen  has  shown,  however,  that  the 
domestic  fowl  is  a  prompt,  reliable  and  liberal  producer  of  precipitins,  even  more 
so  than  the  rabbit.  A  single  intraperitoneal  injection  of  20  c.c.  of  defibrinated 
blood  or  serum  in  most  cases  yields  at  the  end  of  ten  or  twelve  days  a  precipitating 
serum  of  sufficient  strength  and  specificity  for  practical  purposes;  but  on  ac- 
count of  an  unwelcome  tendency  to  give  non-specific  reaction,  great  care  must 
be  exercised  in  all  the  tests  with  fowl  antiserum,  and  it  is  necessary  to  use 
salt  solution  1.8  per  cent,  in  strength.  Man  is  also  a  good  producer  of  precipi- 
tins, as  has  been  shown  by  investigation  of  human  serum  after  the  individual 
has  been  given  doses  of  horse  serum.  For  performing  the  test,  narrow  tubes, 
not  more  than  5  mm.  in  diameter  are  most  suitable  in  order  to  save  reagents 
and  get  clear-cut  results.  Instead  of  diluting  the  antiserum,  it  is  customary 
here  to  dilute  the  antigenic  serum.  Nevertheless  the  titer  thus  obtained  is 
referred  to  the  immune  precipitin.  Two  methods  are  in  use,  the  original 
method  of  actual  precipitation,  and  the  Fornet  ring  test.  In  either  case  dilutions 
of  the  antigenic  serum  are  made  i-io,  i-ioo,  1-1,000,  1-10,000,  1-100,000,  and 
1-1,000,000,  with  provision  for  a  salt  solution  control.  After  such  a  preliminary 
test  the  serum  may  be  more  accurately  titrated  with  intermediate  dilutions.  For 
determining  precipitation  i.o  c.c.  of  each  dilution  is  run  into  tubes  with  a  nipple 
pipette,  and  to  each  is  added  o.i  c.c.  immune  serum,  the  latter  settling  into  the 
dilutions,  without  shaking.  Immediate  observations  are  made  and  then  the 
mixtures  incubated  for  one  hour  at  37°  C,  followed  by  subsequent  observation, 
and  if  desirable  further  observation  after  twenty-four  hours  in  the  ice  chest. 
The  Fornet  ring  test  is  more  clear-cut  and  is  more  commonly  used.  Here  o.i  c.c. 
immune  serum  is  placed  in  the  tubes  and  the  dilutions  of  antigen  added  with 
nipple  pipettes,  so  as  to  form  a  contact  ring  as  in  the  Heller  test  for  albuminuria. 
A  white  ring  gradually  spreading  both  up  and  down  indicates  a  positive  re- 
action. A  good  immune  serum  titrates  1-10,000  or  more,  although  titers  of 
1-100,000  are  obtainable. 

The  production  of  bacterial  precipitins  is  somewhat  more  difficult 
and  requires  longer  immunization,  the  precipitins  appearing,  as  a  rule, 
somewhat  later  than  the  agglutinins.  Zinsser  recommends  the  use  of 
salt  solution  emulsions  of  agar  cultures  killed  at  6o°-7o°  C.,  rather 
than  extracts  or  filtrates  of  broth  cultures.  The  intravenous  route  is 
best  unless  the  bacteria  are  extremely  toxic,  when  the  subcutaneous 
or  intraperitoneal  method  may  serve.  Intravenous  injections  should 
be  given  four  or  five  times  at  five-  or  six-day  intervals,  the  animal 
(rabbit)  being  bled  eight  or  nine  days  after  the  last  injection.  Ex- 
tracts of  bacteria  for  similar  purposes  are  obtained  by  growth  for 
three  weeks  to  three  months  in  slightly  alkaline  broth,  filtration  through 
Berkefeld  filters  and  injection  of  the  filtrate.  Salt  solution  suspen- 
sions of  agar  cultures  may  be  shaken  in  a  machine  for  twenty-four 


AGGLUTININS  AND  PRECIPITINS  109 

hours,  filtered  through  a  Berkefeld  filter  and  the  filtrate  used.  Kraus, 
in  his  original  studies,  used  broth  filtrates  and  also  juice  expressed 
from  the  bacteria.  Kraus  points  out  that  the  broth  filtrates  of  toxin- 
producing  organisms  such  as  bacillus  diphtheriae  do  not  precipitate 
when  mixed  with  antitoxic  serum.  That  this  is  a  general  rule,  how- 
ever, is  not  true,  since  Jacoby  has  shown  that  it  is  possible  to  obtain  a 
precipitate  by  mixing  ricin  and  antiricin  serum,  and  others  have  ob- 
served similar  reaction  with  the  use  of  abrin  and  antiabrin  serum  as 
well  as  crotin  and  anticrotin  serum. 

The  delicacy  of  the  precipitin  reaction  is  great  and  only  exceeded, 
in  certain  respects,  by  complement  fixation  and  the  anaphylaxis  reac- 
tion. It  is  of  interest  to  note  that  whereas  the  Biuret  and  the  Millon 
test  for  protein  will  hardly  exceed  dilutions  of  i-iooo,  the  precipitin 
reaction  will  detect  not  only  the  presence  of  protein  but  the  species 
from  which  it  originates,  commonly  in  dilutions  of  1-10,000  or  1-20,000 
and  even  1-100,000. 

Physical  Basis  of  Precipitation. — The  influence  of  heat  on  pre- 
cipitation and  also  the  group  reactions  are  of  considerable  importance  in 
the  practical  application  of  the  phenomenon  and  will  be  dealt  with  more 
fully  as  this  side  of  the  question  is  considered.  The  comparisons  offered 
between  agglutination  and  certain  colloidal  phenomena  (see  page  94) 
are  equally  applicable  to  precipitation  and  require  no  extensive  dis- 
cussion here.  It  must  be  borne  in  mind,  however,  that  the  colloidal 
interpretation  of  these  phenomena  is  not  proven.  Essentially  the  same 
arguments  are  available  against  the  conception  of  precipitoids  as 
against  that  of  agglutinoids,  but  none  of  these  explains  satisfactorily 
the  specific  absorptive  capacities  of  these  hypothetical  bodies.  As  ag- 
glutinogen  and  agglutinin  may  exist  in  the  blood  of  a  living  animal, 
so  may  precipitinogen  and  precipitin  coexist.  This  is  compared  by 
Zinsser  to  the  fact  that  if  gum  arabic  is  added  to  a  mixture  of  thin 
gelatin  and  arsenic  trisulphide  the  precipitation  which  ordinarily  occurs 
will  be  prevented.  The  gum  arabic  in  this  instance  is  a  protective 
colloid.  It  is  assumed  that  such  a  protective  colloidal  action  operates 
to  prevent  precipitation  when  precipitinogen  and  precipitin  coexist  in 
the  blood  of  a  living  animal.  After  the  blood  is  withdrawn  and 
allowed  to  stand,  this  protective  action  disappears  and  precipita- 
tion occurs. 

The  fact  that  precipitin  and  precipitinogen  can  coexist  in  circulating 
blood  and  that  experiments  on  the  attempted  production  of  iso- 
agglutinins  with  their  conflicting  results  has  led  to  the  question  of 
whether  or  not  it  is  possible  to  produce  precipitins  in  an  animal  by 
the  injection  of  proteins  of  a  closely  related  species.  Uhlenhuth  and 
Weidanz  claim  to  have  produced  precipitins  for  human  serum  by 
injecting  human  serum  into  monkeys,  the  resulting  precipitin  acting 
on  human  but  not  on  monkey  serum.  Berkeley  and  later  Sutherland 
were  unable  to  confirm  this  experiment  and  we  are  forced  to  the  con- 
clusion that  precipitin  formation  in  closely  related  species  is  by  no 
means  a  constant  phenomenon.  Such  precipitins  would  be  practically 


no  THE  PRINCIPLES  OF  IMMUNOLOGY 

iso-precipitins,   and,    as    we   have    seen,   their   existence   is    irregular 
and  questionable. 

Practical  Application. — Wladimiroff  first  applied  precipitation 
practically  in  the  diagnosis  of  glanders  in  horses,  using  the  serum  of 
suspected  horses  against  a  filtrate  from  cultures  of  glanders  bacilli. 
Kraus  employed  the  reaction  to  identify  closely-related  bacteria.  At 
the  present  time,  however,  agglutination  is  employed  for  the  detection 
of  glanders  and  also  for  identification  of  bacteria  rather  than  precipita- 
tion, because  the  latter  procedure  introduces  the  more  cumbersome 
technic  of  obtaining  filtrates. 

The  Forensic  Blood  Test. — Uhlenhuth  and  Beumer  published  their  first  re- 
sults on  the  use  of  the  precipitin  reaction  in  legal  medicine  in  1903.  Other 
studies  were  rapidly  contributed,  and  to-day  the  method  has  an  established  place 
in  the  identification  of  stains  by  blood  and  other  fluids  such  as  seminal  fluid. 
If  spots  on  clothing  or  other  material  are  suspected  of  being  blood,  this  must 
be  proven  by  chemical,  microscopic  or  spectroscopic  examination.  Subsequently 
the  precipitin  test  is  used  to  determine  the  species  from  which  the  blood  orig- 
inated. Before  proceeding  to  this  test  it  is  necessary  to  have  immune  pre- 
cipitating serum  against  the  suspected  species,  usually  man,  an  additional 
immune  precipitating  serum  against  some  other  species  and  a  normal  rabbit  serum. 
The  immune  sera  are  prepared  according  to  the  method  outlined  on  page  108. 
The  suspected  material  must  be  carefully  guarded  against  possible  substitution 
or  contamination  until  the  immune  sera  are  prepared.  It  is  then  dissolved  in 
physiological  salt  solution  and  a  perfectly  clear  filtrate  used.  If  the  material  is 
on  cloth  the  latter  should  be  teased  so  as  to  permit  of  solution ;  if  on  some  solid 
material,  such  as  a  knife  blade,  it  should  be  scraped  off,  ground  in  a  mortar  and 
a  small  amount  of  salt  solution  added.  Cloth  should  be  placed  in  a  test-tube  or 
bottle,  and  it  is  well  to  have  a  control  with  unstained  cloth.  The  time  for 
extraction  depends  to  a  certain  extent  on  the  freshness  of  the  material,  but  it 
is  wise  to  allow  it  to  extract  in  the  refrigerator  over  night,  adding  a  few  drops 
of  chloroform  to  prevent  bacterial  growth.  If  extraction  does  not  proceed  well 
in  salt  solution  it  may  be  necessary  to  extract  with  I  per  cent,  potassium  cyanide 
solution,  correcting  the  alkalinity  after  extraction,  by  means  of  tartaric  acid. 
To  prove  that  the  solution  contains  protein  a  small  amount  may  be  boiled  and 
treated  with  acetic  or  nitric  acid  as  in  the  ordinary  test  for  albuminuria.  A 
final  solution  of  the  suspected  material  in  a  dilution  of  i-iooo  is  usually  emr 
ployed,  and  this  dilution  may  be  approximately  determined  by  the  foam  test. 
For  this  purpose  make  a  i-iooo  solution  of  any  convenient  serum,  blow  air 
through  it  in  a  test-tube  and  note  the  persistence  of  bubbles  above  the  fluid. 
Dilute  the  extract  gradually  and  blow  air  through  it,  repeating  until  that  dilution 
is  obtained  which  will  produce  a  foam  of  about  the  same  viscosity  as  that  in 
the  control  tube.  If  the  solution  is  not  perfectly  clear  it  may  be  centrifuged  or 
passed  through  a  filter  of  washed  asbestos  or  cotton.  On  the  assumption  that 
the  spot  is  suspected  of  being  human  blood  the  test  is  set  up  as  f  ollows : 

Tube  Material  Result 

1  Suspected  extract          -f-  anti-human  serum  + 

2  Suspected  extract          4-  normal  rabbit  serum 

3  Control  extract  4-  anti-human  serum 

4  NaCl  solution  -{-  anti-human  serum 

5  Human  serum  (i-iooo)-f-  anti-human  serum  -f 

6  Beef  serum  (i-iooo)    -f-  anti-human  serum 

7  Sheep  serum  (i-iooo)  -f-  anti-human  serum 

The  amounts  throughout  are  o.i  c.c.  of  each  reagent,  and  the  test  is  made 
by  the  Fornet  ring  method.  Inasmuch  as  acceptable  antisera  should  titrate 
1-10,000,  the  reaction  in  i-iooo  dilution  occurs  within  a  few  minutes,  and  the 
above  result  would  be  interpreted  as  a  clear-cut  positive  for  human  blood  pro- 
vided the  preceding  chemical  and  other  tests  for  blood  had  been  positive.  It 
should  be  remembered  that  the  precipitin  reaction  in  this  instance  simply  iden- 
tifies the  material  as  human  protein,  and  a  similar  result  might  be  obtained 
from  human  seminal  fluid,  albuminous  urine,  purulent  sputum,  exudates  and 
transudates,  unless  the  preliminary  tests  had  been  carried  out. 


AGGLUTININS  AND  PRECIPITINS  in 

Biological  Relationships. — The  question  of  the  specificity  of  this 
reaction  has  been  somewhat  confused  by  quotation  from  the  famous 
studies  of  Nuttall  in  regard  to  interrelationship  of  species.  Nuttall's 
book,  published  in  1904,  wasi  of  the  utmost  importance  to  biology  in 
general,  because  it  demonstrated  anew  by  the  use  of  the  precipitin 
reaction  the  interrelationship  of  animal  species.  He  showed,  for  ex- 
ample, the  close  biological  relationships  between  man  and  the  higher  ape, 
also  similar  relationships  in  the  lower  animals,  as  between  the  goat 
and  the  sheep,  the  horse  and  the  ass.  Reference  to  the  tables  which 
he  published  would  seem  to  indicate,  however,  that  the  relationship 
between  man  and  the  higher  ape  was  so  close  as  to  be  indistinguishable 
by  the  precipitin  reaction.  An  example  in  point  is  the  statement  which 
he  makes  that  whereas  human  blood  will  respond  to  antihuman  precipi- 
tating serum  to  the  extent  of  100  per  cent.,  the  blood  of  the  chimpanzee 
responds  to  the  extent  of  130  per  cent.  These  figures,  however,  refer 
to  the  bulk  of  the  precipitate  thrown  down  in  relation  to  standard 
dilution  of  the  various  bloods  employed.  He  used  relatively  low  dilu- 
tions, allowed  the  sera  to  remain  in  contact  for  several  hours  and  then 
measured  the  amount  of  precipitate.  This,  as  can  readily  be  seen,  is 
different  from  the  method  which  is  employed  at  the  present  time  in 
determining  the  titer  of  the  sera  against  the  immune  serum.  The 
latter  method  is  distinctly  more  delicate  in  determining  the  specificity 
of  the  reaction.  For  that  reason  it  is  the  method  employed  in  the  for- 
ensic test  of  to-day,  as  well  as  in  ordinary  laboratory  procedures.  Fur- 
thermore, we  find  at  the  present  time  that  the  test  demonstrates  its 
specificity  particularly  in  the  presence  of  strong  sera  by  reading  very 
shortly  after  the  contact  has  been  made.  Hektoen  offers  an  excellent 
example  of  this  in  the  following  table  (antihuman  serum)  : 


Blood 

Fish    

Chicken  . . 
Rabbit  ... 
Guinea-pig 

Rat    

Cat    

Dog    

Swine 

Sheep   

Beef    

Horse  

Goat    . 


Monkey   (Macacus  rhesus) 
Human    


-10 

-10 


-10 

-IO 
-IO 
-IO 
-10 
-10 
-IO 
-IO 
-10 
-TOO 

-5000 


It  will  be  noticed  by  reference  to  the  above  table  that  the  titer  of 
the  serum  used  in  this  particular  test  was  only  1-5000,  and  we  would 
expect  an  even  greater  difference  between  the  titer  with  the  different 
animal  sera  if  the  antihuman  serum  had  been  of  higher  titer.  Con- 
cerning the  group  reactions  in  the  precipitin  test  an  interesting  instance 
is  given  by  Hamburger  in  regard  to  the  action  of  the  serum  of  a 
rabbit  inoculated  simultaneously  with  sheep  serum,  goat  serum  and  ox 
serum,  all  of  which  are  fairly  closely  related  to  each  other  biologically. 


ii2  THE  PRINCIPLES  OF  IMMUNOLOGY 

The  serum  of  the  rabbit  when  mixed  separately  with  each  of  these 
three  antigenic  sera  gave  the  most  voluminous  precipitates  in  the 
presence  of  the  sheep  serum,  less  in  the  presence  of  the  goat  serum 
and  least  in  the  presence  of  the  ox  serum.  This  observation  has  been 
confirmed  by  Arrhenius.  Just  why  the  sheep  antiserum  should  be 
the  most  powerful  is  difficult  to  say,  but  it  might  be  assumed  that  the 
sera  of  closely-related  species  may  augment  the  antigenic  power  of 
the  strongest  of  the  three  species  used.  Of  further  interest,  Hektoen 
has  shown  that  in  rabbits  previously  injected  with  foreign  serum  the 
subsequent  injection  of  a  different  serum  may  reawaken  the  production 
of  precipitin  for  the  antigen  previously  injected.  The  practical  value 
of  this  fact  is  that  rabbits  which  have  once  been  used  for  the  production 
of  precipitin  should  not  be  used  again  for  the  same  purpose  with 
another  protein  because  of  possible  decrease  in  specificity  of  the 
second  antiserum. 

Organ  Specificity. — The  question  of  organ  specificity  is  of  con- 
siderable importance  in  the  discussion  of  the  specificity  of  the  precipitin 
test.  Numerous  experiments  have  been  made  by  various  immunological 
methods  to  determine  whether  or  not  it  is  possible  to  identify  the 
protein  of  a  given  organ  within  the  same  species.  It  may  be  stated 
very  briefly  that  these  experiments  have  not  met  with  any  great  degree 
of  success.  However,  in  regard  to  the  protein  of  the  crystalline  lens 
of  the  eye  and  the  protein  of  the  testicle,  certain  interesting  facts  have 
been  discovered.  Immunization  with  protein  extracts  of  the  crystalline 
lens  will  produce  precipitating  sera  which  operate  not  only  on  the 
lens  protein  of  the  same  species  but  on  the  lens  protein  of  all  animals 
as  low  in  order  as  fish.  In  this  example  the  species  specificity  has  been 
entirely  replaced  by  a  curious  organ  specificity.  The  organ  specificity 
in  this  case  is  so  strict  that  the  immune  serum  will  not  react  with  other 
tissue  extract  even  of  the  same  species.  Lens  protein  may,  indeed,  be 
injected  into  the  same  species  from  which  the  lens  was  taken  and  give 
rise  to  specific  precipitins.  By  the  use  of  the  complement-fixation  test 
it  has  further  been  shown  that  in  adult  human  beings  it  is  possible  to 
detect  the  presence  of  an  antibody  for  lens  protein  which  is  not  detect- 
able in  children.  This  phenomenon  will  be  mentioned  later  in  con- 
nection with  the  autocytotoxins  (see  page  142).  Zinsser  comments  to 
the  effect  that  biologically  these  phenomena  probably  signify  that  al- 
though there  are  fundamental  species  differences  between  the  general 
body  proteins  of  various  animals,  there  are  still  in  certain  highly  spe- 
cialized organs  varieties  of  protein  which  possibly  because  of  functional 
exigencies  have  developed  similar  chemical  characteristics.  In  regard 
to  the  testicle  and  the  placenta,  it  might  be  supposed  that  the  germ  char- 
acter of  these  tissues  is  retained  as  distinctive  from  the  somatic  char- 
acter of  the  other  body  tissues.  This  would  not  apply  to  crystalline 
lens,  since  it  is  not  of  germ  character.  On  the  other  hand,  although 
the  lens  can  be  regarded  as  a  highly-specialized  organ  in  both  morpho- 
logical and  physiological  senses,  the  testicle  and  the  placenta  can 
hardly  be  so  considered.  Such  discussions  are  likely  to  be  fruitless 


AGGLUTININS  AND  PRECIPITINS  113 

until  it  is  possible  to  isolate  the  protein  of  other  body  organs  without 
contamination  by  the  animal's  blood.  Up  to  the  present  time  this 
seems  to  be  impossible.  Studies  by  Bell,  for  example,  with  perfusion 
of  various  organs  has  demonstrated  the  impossibility  of  removing  the 
blood  completely. 

Detection  of  Food  Adulteration. — The  precipitin  reaction  is  ap- 
plied not  only  to  detect  blood  as  indicated  above  but  also  various  other 
.body  proteins;  for  example,  it  may  be  used  to  detect  the  nature  of 
bone  fragments  or  other  tissue  scraps.  Of  great  significance  is  the 
fact  that  the  precipitin  test  is  employed  for  the  detection  of  adultera- 
tion of  food  products.  It  has  been  utilized,  for  example,  in  detecting 
adulteration  of  sausages  by  the  use  of  horse  and  other  meats.  In  the 
preparation  of  such  food  products,  heat  is  often  employed,  and  there- 
fore it  is  necessary  to  know  the  influence  of  heat  on  the  precipitin 
reaction.  The  relation  of  heat  to  the  agglutinin  reaction  has  already 
been  discussed  (see  page  93),  and  it  is  found  that  similar  conditions 
exist  in  regard  to  the  precipitin  test.  Obermeier  and  Pick  studied  this 
problem  experimentally  and  found  that  an  antiserum,  even  of  high  titer, 
produced  by  an  unheated  antigen,  failed  to  precipitate  when  brought 
into  contact  with  heated  serum.  If,  however,  animals  are  immunized 
with  serum  boiled  for  a  short  time,  the  resulting  immune  serum  forms 
a  precipitate  when  brought  in  contact  with  either  heated  serum  or 
unheated  serum.  Therefore,  the  precipitin  produced  by  the  latter 
method  is  regarded  as  more  comprehensive  in  its  precipitating  activity, 
but  nevertheless  its  species  specificity  remains  unimpaired.  By  em- 
ploying a  lower  degree  of  heat,  namely  70°  C,  Schmidt  found  that 
this  marked  difference  was  not  so  apparent  and  that  an  immune  serum 
prepared  by  injecting  unheated  serum  would  produce  precipitation 
with  unheated  serum  and  with  the  moderately-heated  serum.  How- 
ever, the  titer  of  antiserum  prepared  by  the  use  of  moderately-heated 
antigen  was  not  as  high  as  with  the  use  of  unheated  antigen.  Schmidt 
ifurther  found  that  he  could  produce  an  even  more  comprehensive 
immune  serum  by  boiling  the  antigen  until  a  coagulum  was  formed, 
namely,  for  three  hours.  The  coagulum  was  washed  with  salt  solution, 
dried,  powdered  and  then  taken  up  with  a  normal  NaOH  solution. 
Zinsser  and  Ottenberg  found  that  the  use  of  a  boiled  antigen  led  to 
the  production  of  a  comprehensive  precipitin,  but  nevertheless  they 
determined  that  this  resulted  in  some  loss  of  specificity  of  the  precipitin. 

This  outline  of  the  influence  of  heat  will  serve  to  show  that  in  the 
detection  of  the  adulteration  of  food  products  extreme  care  must  be 
taken  in  the  selection  of  material.  Wherever  possible,  fresh  material 
should  be  obtained,  and  the  material  for  testing  should  always  be  taken 
from  near  the  middle  of  the  specimen.  This  precaution  prevents  con- 
tamination with  other  meat,  and  in  the  case  of  sausage  yields  material 
likely  to  be  less  influenced  by  heat  or  smoke.  The  meat  is  cut  into  fine 
pieces  and  allowed  to  extract  in  salt  solution.  Clarke  used  30  grams 
meat  and  50  c.c.  physiological  saline,  extracting  in  the  ice  chest  for 
twenty-four  hours,  and  further  diluting  1-300  for  the  test.  Such 
8 


ii4  THE  PRINCIPLES  OF  IMMUNOLOGY 

extracts  must  be  proven  as  to  protein  content  by  the  nitric  acid  test  and 
the  foam  test.  Violent  shaking  is  to  be  avoided,  because  it  liberates 
fats  and  lipoids  which  cloud  the  extract.  If  precipitation  occurs  by  the 
use  of  this  extract  with  an  immune  serum  prepared  by  injecting  un- 
heated  protein,  the  test  can  be  regarded  as  highly  specific.  If,  however, 
it  is  necessary  to  use  serum  which  is  prepared  by  injecting  heated  pro- 
tein, the  specificity  cannot  be  regarded  as  being  so  high.  In  practice 
it  is  the  rule  to  use  serum  prepared  by  injecting  unheated  protein  rather 
than  otherwise,  unless  the  special  indications  of  the  case  indicate  the 
use  of  an  immune  serum  prepared  from  heated  antigen.  The  technic 
in  case  of  food  adulteration  is.  essentially  the  same  as  for  the  detection 
of  blood.  Inasmuch  as  the  specificity  of  this  reaction  is  a  species  speci- 
ficity, it  is  satisfactory  to  utilize  the  animal's  serum  for  immunization 
rather  than  extracts  of  the  flesh  under  suspicion. 

In  mixtures  of  meat  such  as  one  finds  in  sausages,  the  mixture  in 
itself  sometimes  interferes  with  the  delicacy  of  the  test.  In  these 
cases  it  has  been  found  that  the  complement-fixation  test  is  likely  to 
give  more  satisfactory  results. 

The  precipitin  test  is  also  applied  in  the  enforcement  of  game 
laws.  For  example,  cases  arise  in  which  the  unlawful  possession 
of  venison  is  suspected,  and  the  identity  of  the  meat  may  be  estab- 
lished by  the  precipitin  reaction. 

Numerous  suggestions  have  been  made  regarding1  the  identification 
of  racial  strains  within  species,  but  we  agree  with  Hektoen  in  saying 
that  "suggestion  to  the  contrary  notwithstanding,  it  is  not  possible 
to  distinguish  between  different  human  races,  and  far  less  between 
individuals,  by  means  of  the  precipitin  test." 

Function  of  Precipitation  in  Immunity. — The  function  of  pre- 
cipitation in  the  protection  against  infection  is  not  clear,  and,  indeed, 
according  to  certain  theories,  it  may  play  a  part  in  hypersusceptibility. 
Friedberger  has  shown  that  the  addition  of  complement  to  a  precipitin- 
precipitinogen  mixture  leads  to  the  formation  of  a  toxic  body,  but  there 
is  no  convincing  evidence  that  this  actually  takes  place  in  the  living 
animal  (see  page  218) .  It  is  to  be  considered  possible,  on  the  other  hand, 
that  a  certain  amount  of  protection  against  foreign  proteins  may  depend 
on  precipitation,  the  precipitate  being  less  harmful  and  more  suscep- 
tible to  the  destructive  action  of  ferments. 


CHAPTER  VI 
CYTOLYSINS 

INTRODUCTION. 
HEMOLYSINS. 

IMMUNE   HETERO-HEMOLYSINS. 
HEMOLYTIC  AMBOCEPTORS. 

PREPARATION   OF  IMMUNE   HEMOLYSINS. 
OBTAINING   ANTIGENIC   BLOOD. 

PREPARATION  AND  COLLECTION   OF  IMMUNE  SERA. 
TITRATION  OF  IMMUNE  SERA. 
TITRATION  OF  COMPLEMENT. 

QUANTITATIVE  RELATIONS  OF  AMBOCEPTOR  AND  COMPLEMENT. 
QUANTITATIVE  RELATIONS   OF  AMBOCEPTOR   AND  ANTIGEN. 
RELATIVE  AFFINITIES  OF  AMBOCEPTOR  AND  COMPLEMENT. 

SELECTIVE  ABSORPTION  OF  AMBOCEPTOR. 

INFLUENCE  OF  AMOUNT  OF  COMPLEMENT. 

RATE  OF  ABSORPTION  OF  AMBOCEPTOR. 

DISSOCIATION  OF  AMBOCEPTOR  ANTIGEN  UNION. 

SPECIFICITY  OF  AMBOCEPTORS. 

GROUP  REACTIONS. 
NATURE  OF  THE  ANTIGEN. 
NATURE  OF  THE  AMBOCEPTOR. 

MECHANISM  OF  OPERATION  OF  AMBOCEPTOR. 

CONGLUTININS. 
COMPLEMENT. 

DISTRIBUTION. 

ALTERATIONS  OF  AMOUNT. 

METHOD  OF  OBTAINING  COMPLEMENT. 
ORIGIN  OF  COMPLEMENT. 
NATURE  OF  COMPLEMENT. 

PRESERVATION. 

VARIABILITY  OF  COMPLEMENT. 

MULTIPLICITY  OF  COMPLEMENTS. 

COMPLEMENTOIDS. 

COMPLEMENT  FRACTIONS. 

NORMAL    HETERO-HEMOLYSINS. 

PROPORTIONS  OF  AMBOCEPTOR  AND  COMPLEMENT. 
NORMAL  ISO-HEMOLYSINS. 
A  NTI-A  M  BOCEPTORS . 
ANTI-COMPLEMENTS. 
PHYSICAL  HEMOLYSIS. 

FRAGILITY  OF  ERYTHROCYTES. 
CHEMICAL  HEMOLYSIS. 
BACTERIAL  HEMOLYSINS. 

OTHER  VEGETABLE  HEMOLYSINS. 
'     VENOM  HEMOLYSINS. 
CYTOTOXINS. 
SPECIFICITY. 
LENS  CYTOTOXIN. 
BACTERIOLYSINS. 

THE  PFEIFFER  PHENOMENON. 
BACTERIOLYSIS  IN  VITRO. 
WRIGHT'S  METHODS. 
NEISSER-WECHSBERG  PHENOMENON. 
BUXTON'S  METHOD. 
BIOSCOPIC  METHOD. 

SUMMARY. 


n6  THE  PRINCIPLES  OF  IMMUNOLOGY 

Introduction. — In  the  study  of  resistance  to  disease  it  was  learned 
very  early  in  the  course  of  the  investigations  that  the  blood  serum 
possesses  the  property  of  destroying  bacteria.  Later  it  was  found  that 
blood  serum  may  possess  similar  power  in  regard  to  other  cells,  in- 
cluding various  animal  cells,  particularly  the  erythrocytes.  Rather 
than  consider  the  subject  of  cytolysis  in  a  historical  fashion,  we  believe 
that  it  may  be  much  more  clearly  discussed  by  first  presenting  the 
established  facts  which  have  been  learned  concerning  the  power  of 
blood  serum  to  destroy  red  blood-corpuscles.  Many  substances  other 
than  blood  serum  may  destroy  erythrocytes  and  immunology  has 
profited  from  the  study  of  hemolysis  resulting  from  chemical  and 
physical  agents,  but  the  greatest  advance  has  been  made  by  the  investi- 
gation of  the  hemolytic  properties  of  the  blood. 

Hemolysins. — Hemolysins  are  classified,  in  the  same  manner  as 
hemagglutinins,  into  autohemolysins,  iso-hemolysins  and  hetero- 
hemolysins.  These  may  be  present  normally  or  may  be  produced  as  a 
result  of  immunization.  Landois  in  1875  studied  those  normal  hetero- 
hemolysins  which  for  years  have  made  blood  transfusion  a  dangerous 
operation  and  showed  that  fresh  sera  from  various  species  possess  the 
power  of  dissolving  or  laking  the  erythrocytes  of  certain  foreign  species. 
In  1898  Belfanti  and  Carbone  noticed  that  the  serum  of  a  horse  which 
had  received  numerous  injections  of  rabbit  blood  was,  upon  injection, 
specifically  toxic  for  rabbits,  but  they  did  not  determine  the  cause  of 
the  toxicity.  In  the  same  year  Bordet  published  his  discovery  of  the 
fact  that  several  injections  of  defibrinated  rabbit  blood  into  the  peri- 
toneal cavity  of  guinea-pigs  led  to  the  production  of  an  immune  body  in 
the  guinea-pig  serum  capable  of  rapidly  laking  rabbit  erythrocytes, 
whereas  normal  guinea-pig  serum  possesses  the  same  property  in  only 
slight  degree  or  not  at  all.  Shortly  afterward  von  Dungern  and  Land- 
steiner  independently  published  similar  results.  The  immune  body 
in  the  serum  has  been  named  hemolysin  and  also  hemotoxin,  but  the 
former  term  has  received  much  wider  usage,  because  the  constitution 
of  this  body  is  not  that  of  true  toxins,  because  the  effect  is  seen  on  the 
blood-corpuscles  rather  than  the  whole  blood,  and  because  the  hemo- 
globin is  liberated  for  solution  in  the  surrounding  medium  without 
actual  destruction  of  the  stroma.  Bordet  in  his  study  of  the  subject 
showed  that  heating  the  serum  to  55°  C.  for  thirty  minutes  so  altered 
it  that  it  no  longer  produced  hemolysis;  in  other  words,  it  became 
"  inactive."  It  could,  however,  be  reactivated  by  the  addition  of  a 
small  amount  of  fresh  normal  serum.  This  indicated  that  two  sub- 
stances are  concerned  in  the  hemolytic  activity  of  blood  serum,  a 
thermostable  substance  present  in  the  immune  blood  and  a  thermolabile 
substance  present  in  normal  blood  as  well  as  in  immune  blood.  Bordet 
named  the  immune  thermostable  body  "  substance  sensibilisatrice  "  and 
Buchner  named  the  thermolabile  body  "  alexine."  Ehrlich  and  Morgen- 
roth,  whose  studies  have  been  of  fundamental  importance,  named  the 
thermostable  body  "  amboceptor "  and  the  thermolabile  body  "  com- 
plement." Others  have  given  other  names,  but  these  two  forms  of 


CYTOLYSINS  117 

nomenclature  are  most  frequent  in  the  literature.  We  have  elected  to 
use  the  terms  amboceptor  and  complement  because  of  our  belief  that 
these  terms  have  attained  the  more  widespread  usage.  Complement 
appears  in  the  blood  of  many  species,  but  may  be  very  small  in  amount 
or  absent  from  certain  species.  Certain  complements  may  operate 
with  the  amboceptors  of  only  a  few  species,  whereas  others  may  act 
with  amboceptors  of  a  large  number  of  species.  Within  a  given  species 
different  individuals  may  possess  complement  in  variable  quantity,  and 
it  may  vary  at  different  times  in  the  same  individual.  The  complement 
of  guinea-pig  blood  is  usually  large  in  amount  and  applicable  to  the 
amboceptors  of  a  considerable  number  of  other  species.  Complement 
does  not  appreciably  change  in  amount  by  the  ordinary  processes 
of  immunization. 

Immune  Hetero-hemolysins. — Hemolytic  amboceptors  may  be 
natural  to  a  blood  or  may  be  developed  by  immunization.  Autolysins 
and  isolysins  may  be  produced  but  with  great  difficulty  and  variability. 
Isolysins  may  be  present  normally,  notably  in  man.  Heterolysins  may 
be  found  normally  and  can  be  readily  produced  by  artificial  immuniza- 
tion. Bordet  produced  heterolysins  by  intraperitoneal  injection  of  ery- 
throcytes.  They  may  also  be  induced  by  the  subcutaneous  and  by  the 
intravenous  routes  of  inoculation.  Two  important  conceptions  of  the 
mode  of  action  of  the  amboceptor  have  been  proposed.  Bordet,  Metch- 
nikoff  and  the  French  school  consider  the  action  to  be  in  the  nature  of  a 
sensitization  or  fixation  of  the  antigenic  cells,  so  that  they  are  more 
readily  acted  on  by  the  complement,  in  somewhat  the  same  fashion 
that  a  mordant  prepares  a  cell  so  that  it  will  stain  more  readily.  Ehr- 
lich  and  Morgenroth  and  the  German  school  consider  the  amboceptor 
as  a  link  which  brings  together  antigen  and  complement ;  in  other  words, 
in  their  conception  the  amboceptor  possesses  two  binding  groups,  a 
cytophilic  and  a  complementophilic  group,  each  capable  of  acting  as  a 
specific  receptor.  In  order  to  discuss  the  various  theoretical  con- 
siderations more  clearly,  it  is  essential  that  the  well-established  facts  in 
regard  to  hemolysis  be  presented  as  they  are  ordinarily  demonstrated 
in  a  practical  way. 

Preparation  of  Immune  Hemolysins.  The  Blood  Antigen. — In  immuniza- 
tion for  the  production  of  a  hemolysin  it  is  necessary  to  select  the  animals  to 
be  used.  The  rabbit  is  usually  chosen  as  the  animal  to  be  immunized  because 
of  the  fact  that  it  is  easily  available,  relatively  inexpensive,  and  yields  a  fairly 
large  amount  of  blood.  In  selecting  the  animal  whose  blood-corpuscles  are  to 
be  used  for  the  production  of  hemolysin,  convenience  again  plays  a  part.  The 
sheep  is  the  animal  most  commonly  employed,  although  the  goat  is  equally  use- 
ful. Reasonably  large  amounts  of  blood  can  be  secured  from  such  animals  at 
short  intervals  of  time,  without  deleterious  effects.  Dog  blood  is  unsatisfactory 
because  the  corpuscles  do  not  resist  standing  for  any  length  of  time.  The  cat  is 
undesirable  because  of  its  relatively  small  size.  In  order  to  secure  blood  from 
a  goat  or  sheep  the  animal  is  either  strapped  on  a  board,  or  may  be  held  by  a 
skilled  attendant.  The  neck  is  shaved  over  the  jugular  vein,  the  area  washed 
with  soap  and  water,  cleansed  with  alcohol,  and  the  vein  distended  by  pressure 
over  the  jugular  bulb  at  the  base  of  the  neck.  The  blood  is  collected  through  a 
fairly  large  needle  into  a  sterile  flask  containing  glass  beads  or  fragments  of 
glass  tubing.  Rotation  of  the  flask  or  shaking  during  the  collection  and  con- 
tinued shaking  for  five  or  ten  minutes  after  the  collection  completely  defibrinates 


ii8  THE  PRINCIPLES  OF  IMMUNOLOGY 

the  blood.  The  blood  may  be  injected  as  defibrinated  blood  for  purposes  of  im- 
munization, but  as  a  rule  in  order  to  avoid  any  influences  the  serum  may  have, 
the  blood  is  washed  so  that  only  corpuscles  are  injected.  For  purposes  of  washing 
50  c.c.  centrifuge  tubes  are  desirable,  but  if  these  are  not  obtainable,  the  15  c.c. 
size  may  be  employed.  The  blood  is  measured  into  the  tube  with  a  pipette, 
usually  to  the  amount  of  5.0  c.c.  The  amount  of  blood  is  marked  with  a 
grease  pencil  and  the  tube  filled  with  physiologic  salt  solution.  The  tube  is 
centrifuged  until  the  blood  is  thrown  down.  The  supernatant  fluid  is  poured  off 
and  the  tubes  again  filled  with  salt  solution.  The  sedimented  corpuscles  are 
shaken  up  into  the  salt  solution  and  again  centrifuged.  This  operation  is  re- 
peated again,  and  the  blood  is  said  to  have  been  washed  three  times.  After 
the  last  centrifugation  the  supernatant  fluid  is  poured  off  and  the  sedimented 
blood-corpuscles  restored  to  original  volume  by  addition  of  salt  solution.  In 
order  to  make  a  five  per  cent,  suspension,  this  is  washed  into  a  100  c.c.  cylinder 
and  made  up  to  100  c.c.  volume  with  salt  solution.  Any  other  percentage 
desirable  may  be  made  by  appropriate  additions  of  salt  solution  to  the  original 
blood  mass. 

Preparation  and  Collection  of  Immune  Sera.—  The  injection  into  the  rabbit 
may  be  by  subcutaneous,  intraperitoneal,  or  intravenous  routes.  The  intravenous 
route  produces  immune  bodies  most  rapidly,  as  has  been  shown  by  Bullock, 
and  as  a  rule  produces  an  immune  serum  of  higher  titer  than  is  obtainable  by 
other  methods.  An  excellent  way  to  produce  hemolysin  rapidly  is  to  inject  in- 
travenously into  the  rabbit  three  doses  4.0  c.c.  each  of  50  per  cent,  suspension 
of  washed  sheep  or  goat  erythrocytes  at  intervals  of  five  days.  The  method  of 
intravenous  injection  has  previously  been  described  in  connection  with  the  pro- 
duction of  agglutinins  (see  page  82).  A  test  bleeding  may  be  made  from  the 
posterior  ear  vein  five  to  seven  days  after  the  last  injection,  and  if  the  titer  of 
the  serum  is  not  sufficiently  high,  one  or  two  more  injections  may  be  given.  When 
sufficiently  high  the  rabbit  is  bled  from  the  femoral  artery  as  previously 
described  (see  page  83).  The  blood  is  collected  in  a  flask,  the  flask  inclined 
at  an  angle  of  about  45°  until  the  blood  is  firmly  clotted.  The  flask  is  then  placed 
in  an  upright  position  in  the  refrigerator  for  about  twenty-four  hours,  after 
which  the  collected  serum  is  pipetted  into  a  sterile  container.  Melick,  in  a  study 
of  the  influence  of  colloidal  suspensions  on  the  production  of  hemolysis,  finds 
that  if  he  gives  preliminary  intravenous  injection  of  aleurpnat  suspension  and 
subsequently  immunizes  with  blood-corpuscles,  the  hemolytic  sera  are  of  con- 
siderably higher  titer  than  in  animals  not  so  treated. 

Titration  of  Immune  Sera.  —  For  titration  of  the  hemolysin  in  the  rabbit 
serum,  it  is  necessary  to  have  a  5  per  cent,  suspension  of  the  antigenic  corpuscles, 
the  serum  to  be  tested  and  in  addition  fresh  guinea-pig  serum  which  serves  as 
complement.  In  such  a  titration  the  5  per  cent,  suspension  of  corpuscles  and  the 
complement  are  regarded  as  standards  and  employed  in  the  same  doses  through- 
out the  series  of  tubes.  If  0.5  c.c.  of  serum  and  0.5  c.c.  of  corpuscle  suspension 
are  employed,  0.05  c.c.  of  complement  is  usually  sufficient,  Before  attempting 
the  titration  the  rabbit  immune  serum  should  be  inactivated  by  heating  in  a 
water  bath  at  56°  C.  for  one-half  hour.  Dilutions  of  the  inactivated  immune 
amboceptor  are  made  as  a  rule  i-io,  i-ioo,  1-500,  i-iooo,  1-1500,  1-2000,  1-2500, 
1-3000,  1-4000.  The  guinea-pig  serum  (complement)  is  diluted  i-io.  The  follow- 
ing protocol  will  show  how  the  series  of  tubes  is  set  up  : 


Amboceptor  Complement  i-io  Result 

0.5  c.c.                    -ioo      0.5  c.c.  0.5  c.c.  CH 

0.5  c.c.                    -500      0.5  c.c.  0.5  c.c.  CH 

0.5  c.c.                    -1000    0.5  c.c.  .          0.5  c.c.  CH 

0.5  c.c.                    -1500    0.5  c.c.  0.5  c.c.  CH 

0.5  c.c.                    -2000    0.5  c.c.  0.5  c.c.  CH 

0.5  c.c.                    -2500    0.5  c.c.  0.5  c.c.  CH 

0.5  c.c.                    -3000    0.5  c.c.  0.5  c.c.  PH 

0.5  c.c.                    -4000    0.5  c.c.  0.5  c.c.  N 

0.5  c.c.                   ......     0.5  c.c.  0.5  c.c.  N 

0.5  c.c.                   i-ioo      0.5  c.c.  ......  N 

The  last  two  tubes  are  controls  and  should  be  made  up  to  volume  by  addi- 
tion of  0.5  c.c.  saline  in  place  of  amboceptor  in  the  one  and  of  complement  in  the 

other.     The  letters  CH  indicate  complete  hemolysis,  PH  partial  hemolysis,  and 


a 


FIG.  12. — a  shows  a  saline  suspension  of  blood-corpuscles   before 

hemolysis;    b   the  same  after  hemolysis.     a'    and   b'   present   the 

appearance  of  a  and  b  after  sedimentation  of  corpuscles.     From 

Noguchi,  Serum  Diagnosis  of  Syphilis. 


Green  -»  Complement 
Purple  <=  Amboceptor 
Red  =•  Haemolysis 


/unit  of  Amboceptor 
used  in  esc/7  w/mvar/ous 
fractions  or  3  comp/ement 
unit 


FIG.  13. — Diagrams  showing  relative  proportions  of  reagents  in  hemolysis.  The 
group  of  four  columns  shows  the  reduction  possible  in  amounts  of_  complement  with 
increasing  amounts  of  hemolytic  amboceptor,  in  the  production  of  complete  hemol- 
ysis. The  second  and  third  parts  of  the  figure  show  the  curve  of  reduction  in 
percentage,  or  degree,  of  hemolysis  following  reduction  in  amount  of  comple- 
ment in  the  presence  of  constant  quantities  of  amboceptor.  From  Noguchi,  Serum 
Diagnosis  of  Syphilis. 


CYTOLYSINS  119 

N  no  hemolysis,  the  reading  being  made  after  a  period  of  incubation  in  the 
water  bath  at  37°  C.  This  period  may  be  thirty  minutes,  one  hour  or  two  hours, 
but  subsequent  experiments  with  the  same  system  of  amboceptor,  complement 
and  corpuscles  must  be  made  with  the  same  period  of  incubation  as  practised  in 
the  original  titration.  In  this  laboratory  one  hour  is  the  standard  time  for  incu- 
bation. In  order  to  make  results  somewhat  more  clear-cut,  the  rack  of '  test 
tubes  may  be  placed  in  the  refrigerator  over  night  and  the  results  read  the  fol- 
lowing morning.  The  lapse  of  twelve  or  eighteen  hours  time  permits  the  cor- 
puscles to  settle  to  the  bottom'  of  the  test-tube ;  therefore  any  red  coloring  of 
the  supernatant  fluid  may  be  interpreted  as  a  partial  or  complete  hemolysis,  de- 
pending on  the  depth  of  color  and  the  amount  of  sediment  remaining  on  the 
bottom  of  the  tube.  The  controls  which  are  used  in  this  experiment  demon- 
trate  that  neither  complement  nor  inactivated  amboceptor  will  produce  hemoly- 
sis. The  result  given  in  the  above  experiment  indicates  that  at  some  point 
between  the  dilutions  1-2500  and  1-3000  the  exact  end  point  of  titration  is  to  be 
found.  In  order  to  determine  the  exact  end  point  it  is  well  to  set  up  an  addi- 
tional series  with  dilutions  of  1-2500,  1-2600,  1-2700,  1-2800,  1-2000,  and  1-3000 
with  the  necessary  controls.  If  it  is  found  that  complete  hemolysis  takes  place 
in  a  dilution  1-2700  and  not  in  the  dilution  1-2800  the  dilution  1,2700  is  taken  as 
the  end  point  or  titer.  The  unit  of  amboceptor  therefore  is  1-2700  of  0.5  c.c.  or 
1-5400  of  i  c.c.  In  the  experiment  outlined  above,  the  unit  of  amboceptor  would 
be  designated  as  0.5  c.c.  of  a  1-2700  dilution  of  the  immune  serum. 

Titration  of  Complement. — As  has  been  indicated  previously,  the  amount 
of  complement  in  guinea-pig  serum  varies  in  different  animals.  Therefore  sub- 
sequent experiments  with  this  amboceptor  must  be  controlled  by  titrating  the 
complement.  This  may  be  done  by  setting  up  a  series  of  tubes  as  follows,  the 
control  tubes  being  made  up  to  volume  with  salt  solution : 

Erythrocytes  Amboceptor  Complement  PPC,,H 

suspension  1-2700  i-io 

0.5  c.c.  0.5  c.c.  0.5  c.c.  CH 

0.5  c.c.  0.5  c.c.  0.4  c.c.  CH 

0.5  c-c.  0.5  c.c.  0.3  c.c.  PH 

0.5  c-c.  0.5  c.c.  0.2  c.c.  N 

0.5  c-c.  0.5  c.c.  o.i  c.c.  N 

0.5  c.c.  ...  0.5  c.c.  N 

0.5  c.c.  0.5  c.c.  ...  N 

0.5  c.c.  ...  ...  N 

In  this  experiment  it  is  found  that  0.4  c.c.  of  the  new  complement  is  sufficient 
for  activating  the  unit  of  amboceptor.  Therefore,  whereas  in  the  first  experiment 
0.5  c.c.  i-io  complement  dilution  was  the  unit  of  complement,  in  the  second  experi- 
ment 0.4  c.c.  i-io  dilution  complement  is  the  unit.  If  it  is  found  that  in  none  of 
these  tubes  complete  hemolysis  takes  place  because  of  weak  complement,  it  will 
then  be  necessary  to  set  up  an  additional  series  with  complement  diluted  1-5 
instead  of  i-io. 

Quantitative  Relations  of  Amboceptor  and  Complement. The 

quantitative  relationship  between  the  amount  of  complement  and  ambo- 
ceptor used  has  been  very  extensively  studied.  It  is  now  known  that 
a  larger  amount  of  complement  will  require  a  smaller  amount  of  ambo- 
ceptor for  the  production  of  complete  hemolysis  in  the  standard  blood- 
corpuscle  suspension  and  conversely  a  smaller  amount  of  complement 
requires  a  larger  amount  of  amboceptor.  Thus,  if  we  use  two  units  of 
complement,  hemolysis  will  occur  in  the  presence  of  less  than  one  unit 
of  amboceptor.  If  we  use  two  units  of  amboceptor,  it  will  require 
less  than  one  unit  complement  to  produce  complete  hemolysis.  This 
relationship,  however,  is  not  in  definite  proportion.  For  example,  if 
four  units  of  amboceptor  are  employed,  one-third  unit  of  complement 
is  necessary.  This  relationship  is  beautifully  illustrated  in  the  dia- 
gram (Fig.  13)  taken  from  Noguchi. 


120  THE  PRINCIPLES  OF  IMMUNOLOGY 

Quantitative  Relations  of  Amboceptor  and  Antigen. — By  subse- 
quent studies  of  different  mixtures,  it  was  found  that  the  unit  of 
standard  corpuscle  suspension  can  take  up  considerably  more  than  one 
unit  of  amboceptor.  This  amount  varies  with  the  total  quantity  of  im- 
mune body  present.  For  example,  Muir  found  that  on  addition  of 
twelve  doses  of  amboceptor  one  dose  remained  free,  on  addition  of  six- 
teen doses  of  amboceptor  two  doses  remained  free,  on  addition  of 
twenty  doses  three  doses  remained  free  and  on  the  addition  of  twenty- 
three  doses  of  amboceptor,  four  doses  remained  free.  When,  how- 
ever, the  mixture  of  complement,  amboceptor  and  red  blood- 
corpuscles  is  properly  adjusted,  the  reaction  completely  uses  up  the 
amboceptor,  complement  and,  by  hemolysis,  all  the  red  blood-corpuscles. 
Correspondingly,  if  two  units  of  complement  are  employed  in  the 
presence  of  one  unit  of  amboceptor,  it  does  not  follow  that  after 
the  reaction  one  unit  of  complement  will  remain  free.  As  a  matter 
of  fact,  practically  the  entire  two  units  of  complement  will  be  utilized 
in  the  reaction.  Nevertheless,  increasing  the  amounts  of  complement 
will  leave  more  and  more  complement  free  in  the  supernatant  fluid. 
These  points  will  be  made  somewhat  clearer  after  subsequent  experi- 
ments have  been  outlined. 

Relative  Affinities  of  Amboceptor  and  Complement. — In  the  in- 
troductory paragraph  it  was  pointed  out  that  the  amboceptor  has  a 
special  affinity  for  the  antigenic  red  blood-corpuscles,  but  that  the  com- 
plement has  no  such  affinity.  This  is  illustrated  by  the  fact  that  when 
red  blood-corpuscles  are  set  up  against  amboceptor  they  will  absorb 
the  amboceptor,  but  if  they  are  set  up  in  the  presence  of  complement 
they  will  not  absorb  complement. 

The  following  experiment  illustrates  this  point:  Two  centrifuge  tubes 
are  marked  A  and  B.  In  tube  A  are  placed  i.o  c.c.  standard  erythrocyte  sus- 
pension (5  per  cent,  suspension)  and  i.o  c.c.  inactivated  immune  serum  so 
diluted  as  to  contain  two  units  amboceptor.  This  tube  is  incubated  at  37°  C. 
for  thirty  minutes  and  then  centrifuged.  The  supernatant  fluid  is  pipetted 
into  a  tube  marked  A  2.  The  erythrocyte  sediment  in  tube  A  is  washed  in 
salt  solution,  again  centrifuged  and  the  supernatant  fluid  discarded.  The 
sediment  in  tube  A  is  resuspended  in  i.o  c.c.  salt  solution  and  two  units  of 
complement,  i.e.,  i.o  c.c.  i-io  dilution  are  added.  To  tube  A  2  are  added  two 
units  complement  (i.o  c.c.  i-io  dilution)  and  i.o  c.c.  5  per  cent,  'erythrocyte 
suspension.  These  tubes  are  incubated  for  one  hour  at  37°  C.  Tube  A  will 
show  hemolysis  because  the  sedimented  corpuscles  have  absorbed  ambo- 
ceptor and  the  addition  of  complement  is  sufficient  to  complete  the  reaction. 
Tube  A  2  will  not  show  hemolysis  because  the  amboceptor  is  not  in  the 
supernatant  fluid  and  the  complement  is  not  sufficient  to  lake  the  added 
corpuscles.  At  the  same  time  the  converse  of  the  foregoing  experiment  may 
be  conducted.  In  tube  B  are  placed  i.o  c.c.  standard  erythrocyte  suspension 
(5  per  cent,  suspension)  and  i.o  c.c.  fresh  guinea-pig  serum  diluted  i-io. 
This  is  incubated  thirty  minutes  at  37°  C,  centrifuged  and  the  sediment 
washed.  The  supernatant  fluid  is  placed  in  tube  B  2.  The  sediment  in  tube  B 
is  resuspended  in  i.o  c.c.  salt  solution  and  i.o  c.c.  immune  serum  so  diluted 
as  to  contain  two  units  amboceptor  is  added.  To  the  supernatant  fluid  in 
tube  B  2  are  added  i.o  c.c.  immune  serum  (two  units  amboceptor)  and  i.o  c.c. 
erythrocyte  suspension.  These  tubes  are  incubated  for  one  hour  at  37°  C. 
Tube  B  2  will  show  hemolysis  because  the  supernatant  fluid  after  the  centrifu- 
gation  still  contains  complement,  so  that  the  addition  of  amboceptor  and 
erytnrocytes  permits  of  completion  of  the  reaction.  Tube  B  will  not  show 


CYTOLYSINS  121 

hemolysis  because  the  corpuscles  in  the  sediment  have  not  taken  up  any 
complement,  and  the  addition  of  amboceptor  is  not  sufficient  for  the 
reaction  to  occur. 

Selective  Absorption  of  Amboceptor  and  Complement. — Not  only 
is  it  possible  to  show,  as  has  been  done  in  the  preceding  experiment, 
that  red  blood-corpuscles  will  combine  with  amboceptor  and  not  with 
complement,  but  if  conditions  are  so  arranged  that  hemolysis  is  pre- 
vented, it  is  possible  to  demonstrate  that  red  blood-corpuscles  will 
selectively  absorb  amboceptor  from  a  mixture  of  amboceptor  and 
complement.  In  order  to  prevent  hemolysis,  it  is  necessary  to  permit 
the  absorption  to  take  place  at  o°  C.  Not  only  must  this  precaution 
be  observed,  but  the  tubes  must  be  cooled,  the  various  reagents  in  the 
mixture  must  be  cooled  in  advance  and  the  centrifuge  carrier  must 
also  be  cooled. 

The  various  reagents  are  placed  in  test  tubes  and  all  the  tubes  placed  in 
a  mixture  of  salt  and  ice.  Into  a  cold  centrifuge  tube  are  placed  i.o  c.c. 
5  per  cent,  erythrocyte  suspension,  i.o  c.c.  inactive  immune  serum  so  diluted 
as  to  contain  two  units  amboceptor  and  i.o  c.c.  guinea-pig  serum,  i-io  dilu- 
tion. This  tube  remains  in  the  salt-ice  mixture  for  thirty  minutes  and  is 
then  centrifuged.  The  supernatant  fluid  is  poured  off  and  divided  so  that 
one-half  the  amount  is  placed  in  each  of  two  tubes.  The  sediment  is  washed 
in  cold  salt  solution  and  resuspended  in  4.0  c.c.  cold  salt  solution.  These 
4.0  c.c.  are  divided  between  two  tubes.  The  four  tubes  so  prepared  are  set 
up  as  follows: 

TUBE  i 

Supernatant  fluid  1.5  c.c. 

Fresh  guinea-pig  serum,  i-io 0.5  c.c. 

5  per  cent,  erythrocyte  suspension 0.5  c.c. 

TUBE  2. 

Supernatant  fluid  1.5  c.c. 

Immune  rabbit  serum 0.5  c.c. 

5  per  cent,  erythrocyte  suspension 0.5  c.c. 

TUBE  3. 

Sediment  2.0  c.c. 

Fresh  guinea-pig  serum,  i-io 0.5  c.c. 

TUBE  4. 

Sediment  2.0  c.c. 

Immune  rabbit  serum 0.5  c.c. 

These  tubes  are  incubated  for  one  hour  at  37°  C.  Inasmuch  as  the 
supernatant  fluid  no  longer  contains  amboceptor,  tube  i  will  fail  to  show 
hemolysis,  but  in  the  case  of  tube  2  the  amboceptor  is  added,  and  since  the 
supernatant  fluid  contains  complement  which  has  not  been  absorbed  by  the 
corpuscles,  hemolysis  will  result.  The  sediment  has  absorbed  amboceptor 
from  the  mixture ;  therefore,  in  the  case  of  tube  3,  the  addition  of  fresh  guinea- 
pig  serum  will  serve  to  produce  hemolysis.  The  sediment  has  not,  however, 
taken  up  any  complement,  the  addition  of  the  immune  serum  in  tube  4  will 
not  serve  to  complete  the  reaction,  and  hemolysis  will  not  occur.  By  the 
use  of  sera  containing  other  hemolytic  amboceptors,  it  is  possible  to  show 
not  only  that  absorption  of  amboceptor  may  occur  from  a  complement 
amboceptor  mixture,  but  that  this  absorption  is  specific  for  the  particular 
amboceptor  concerned. 

Influence  of  Amount  of  Complement. — Although,  as  will  be  shown 
subsequently,  the  concentration  of  complement  plays  a  part  in  the  com- 


122  THE  PRINCIPLES  OF  IMMUNOLOGY 

pletion  of  hemolysis  it  can  be  demonstrated  that  the  absolute  amount 
rather  than  the  degree  of  concentration  is  of  importance  in  regard  to 
the  amboceptor. 

This  may  be  shown  by  placing  in  each  of  four  tubes  0.5  c.c.  5  per  cent, 
suspension  of  corpuscles  and  adding  to  the  second,  third  and  fourth  tubes, 
respectively,  four,  nine  and  fourteen  volumes  of  salt  solution.  To  each  of  the 
four  tubes  is  added  one  unit  amboceptor,  and  the  mixture  incubated  at  37°  C 
for  one-half  hour  to  permit  absorption  of  amboceptor.  The  tubes  are  cen- 
trifuged  and  the  supernatant  fluid  is  discarded.  To  each  tube  is  added  i.o  c.c. 
complement  so  diluted  as  to  contain  one  unit,  and  the  mixtures  again  incu- 
bated at  37°  C.  for  one  hour.  Hemolysis  will  occur  equally  in  all  tubes 
showing  that  the  complete  absorption  of  amboceptor  by  the  cells  occurred 
in  spite  of  marked  dilution  in  some  of  the  tubes. 

Rate  of  Absorption  of  Amboceptor. — The  absorption  of  ambo- 
ceptor varies  in  rapidity  under  different  conditions.  For  example, 
absorption  takes  place  more  readily  at  37°  C.  than  at  20°  C.,  and  more 
readily  at  20°  C.  than  at  o°.  An  exception  to  this  rule  appears  in 
cases  of  paroxysmal  hemoglobinuria.  Some  of  these  cases  possess  in 
the  blood  an  autohemolysin  which  does  not  enter  into  combination  with 
erythrocytes  at  body  temperature.  If  the  blood  is  withdrawn  and 
placed  at  a  temperature  of  o°  to  10°  C.  for  an  hour  the  cells  absorb  the 
amboceptor  and  subsequent  incubation  at  37°  C.  permits  the  inter- 
action of  complement  so  that  hemolysis  results.  With  this  and 
possibly  some  other  exceptions  the  general  rule  holds  true  that  tem- 
peratures approaching  37°  C.  favor  the  union  of  amboceptor  and  antigen. 
Certain  physical  conditions  also  play  a  part  in  rapidity  of  absorption 
as  may  be  shown  by  the  following  experiment  in  which  the  mixture  of 
corpuscles  and  amboceptor  is  made  under  different  conditions. 

Two  wide  test  tubes  or  small  beakers  are  marked  A  and  B.  In  A  are 
placed  six  units  of  cell  suspension;  namely,  3.0  c.c.  5  per  cent,  suspension. 
To  this  are  added  drop  by  drop  3.0  c.c.  amboceptor,  so  diluted  that  it  con- 
tains six  units,  the  tube  being  shaken  constantly  during  the  addition.  In 
tube  B  the  process  is  reversed,  the  amboceptor  being  placed  in  the  tube  and 
the  cell  suspension  added  drop  by  drop.  These  mixtures  may  be  titrated 
against  varying  amounts  of  complement  in  a  series  of  tubes,  or  six  units  of 
complement  may  be  added  to  tube  A  and  tube  B.  An  hour's  incubation  at 
37°  C.  will  show  less  active  hemolysis  in  tube  B  than  in  A.  The  probable 
explanation  is  that  the  first  cells  added  to  the  amboceptor  in  tube  B  absorb 
all  or  nearly  all  the  amboceptor,  and  the  subsequently  added  cells  are  only 
partly  saturated  or  take  up  no  amboceptor  at  all. 

This  experiment  illustrates  the  very  rapid  absorption  of  amboceptor 
by  cells  and  also  the  fact  that  cells  may  absorb  considerably  more  than 
one  unit  of  amboceptor. 

Dissociation  of  Amboceptor-Antigen  Union. — Whereas  tempera- 
tures up  to  37°  C.  appear  to  favor  absorption  of  amboceptors,  Bail, 
Tsuda  and  others  have  shown  that  a  temperature  of  42°  C.  results  in 
a  partial  dissociation  of  amboceptor.  That  dissociation  of  amboceptor 
and  cells  could  occur  was  shown  independently  by  Muir  and  by  Mor- 
genroth.  Muir  mixed  i.o  c.c.  5  per  cent,  corpuscle  suspension  with 
ten  units  amboceptor  and  allowed  the  mixture  to  stand  at  room  tem- 
perature for  one  hour.  The  tube  was  then  centrif uged,  the  corpuscles 


CYTOLYSINS  123 

washed  three  times  and  resuspended  in  salt  solution  to  a  volume  of  i.o 
c.c.  To  this  was  added  i  .o  c.c.  untreated  corpuscle  suspension,  the  tube 
shaken  and  placed  at  37°  C.  for  one  hour.  At  the  end  of  this  time 
four  units  of  complement  were  added,  the  tube  incubated  again  for 
one  hour  and  complete  hemolysis  was  found.  Thus  it  was  found  that 
the  original  cells  yielded  at  least  one  unit  of  amboceptor  for  the  new 
cells.  Although  in  the  report  cited  on  page  120,  twelve  units  gave  one 
free  unit,  Muir  states  that  usually  one  unit  of  amboceptor  can  be 
obtained  from  corpuscles  containing  six  units.  In  this  experiment  the 
dissociation  was  at  37°  C.,  but  dissociation  takes  place  at  room  tem- 
perature, although  more  slowly,  and  at  o°  C.  it  is  practically  nil.  By 
working  with  sensitized  cells  and  with  supernatant  fluids,  it  is  possible 
to  titrate  the  latter  so  as  to  determine  the  exact  quantities  of  ambo- 
ceptor dissociated.  Kosakai,  in  working  with  so-called  pure  hemolysins, 
has  recently  shown  that  the  antigen  and  amboceptor  union  is  reversible  to 
a  greater  extent  than  has  previously  been  supposed.  He  main- 
tains that  the  reversibility  under  these  circumstances  is  almost  or 
quite  complete. 

Specificity  of  Amboceptors.  Group  Reactions. — The  hemolytic 
amboceptors  are  highly  specific,  but  show,  as  do  other  immune  bodies, 
group  reactions.  Ehrlich  and  Morgenroth  showed  that  immune  sera 
prepared  against  ox  blood  are  hemolytic  also  for  goat  and  sheep  blood 
and  that  a  hemolysin  prepared  against  goat  blood  also  dissolves  ox 
blood.  Marshall  showed  that  an  antihuman  hemolysin  acts  on  monkey 
blood  and  vice  versa.  In  any  case  the  hemolysin  is  most  active  in  the 
presence  of  the  antigenic  corpuscles.  Treatment  of  a  hemolytic  im- 
mune serum  with  heterologous  corpuscles  removes  more  of  the  specific 
immune  body  than  is  the  case  in  other  group  reactions.  For  example, 
Muir  developed  an  anti-ox-blood  serum  which,  in  a  dose  of  0.0005  c.c. 
dissolved  i.o  c.c.  5  per  cent,  suspension  ox  corpuscles  and,  in  a  dose 
of  0.0012  c.c.  dissolved  a  similar  suspension  of  sheep  corpuscles.  Ab- 
sorption by  sheep  corpuscles  in  excess  reduced  the  titer  against  ox  cor- 
puscles so  that  the  serum  dissolved  the  latter  in  doses  of  0.0012  c.c. ; 
in  other  words,  the  titer  of  the  serum  was  reduced  to  about  half  its 
original  strength,  Ehrlich  and  Morgenroth  showed  that  if  the  quantity 
of  sheep  corpuscles  is  carefully  adjusted  so  as  exactly  to  equal  the 
hemolytic  power  for  such  corpuscles,  the  fraction  of  amboceptor  lytic 
for  sheep  corpuscles  may  be  absorbed  without  reducing  the  titer  against 
ox  cells.  If,  however,  the  amboceptor  for  ox  blood  is  removed  by 
absorption  with  ox  corpuscles  the  hemolytic  power  for  sheep  corpuscles 
is  entirely  destroyed.  Thus  it  is  seen  that  there  is  close  similarity  with 
the  group  reactions  of  agglutinins  and  other  immune  bodies.  Ehrlich 
and  Morgenroth  explain  the  phenomenon  by  assuming  that  each  ambo- 
ceptor contains  numerous  "  partial  amboceptors  "  formed  in  the  im- 
mune animals  in  response  to  relatively  undifferentiated  receptors  of  the 
antigenic  cells.  In  other  words,  ox-blood  corpuscles  are  supposed  to 
contain  a  certain  number  of  receptors  specific  to  those  cells,  and  in 
addition  other  receptors  that  are  closely*  similar  to  or  identical  with 


124  THE  PRINCIPLES  OF  IMMUNOLOGY 

certain  receptors  of  sheep  cells  and  goat  cells.  Therefore,  the  injec- 
tion of  ox  cells  leads  to  the  production  of  an  amboceptor  containing 
partial  amboceptors  specific  for  ox  blood  and  partial  amboceptors 
specific  for  the  common  receptors  of  ox,  sheep  and  goat  cells.  The 
removal  of  the  partial  amboceptors  common  to  all  three  cell  receptors 
will  not  remove  that  specific  for  ox  cells,  but  ox  cells  will  remove  both 
the  specific  and  common  fractions.  This  explanation  has  been  the 
subject  of  much  experiment,  particularly  with  anti-hemolysins,,  and 
modern  views  are  not  entirely  in  accord  with  the  original  views  of 
Ehrlich.  The  subject  will  be  referred  to  again  in  connection  with  a 
discussion  of  anti-amboceptors  and  anti-complements.  In  the  same 
place  will  be  found  a  discussion  of  the  interpretation  of  the  ambo- 
ceptor as  made  up  of  a  cytophilic  and  complementophilic  group. 

Nature  of  the  Antigen. — In  ordinary  practice  the  entire  erythrocyte 
is  employed  for  immunization,  but  attempts  have  been  made  to  deter- 
mine what  fraction  of  the  cell  is  truly  antigenic.  Ford  and  Halsey 
have  shown  that  the  use  of  either  stroma  or  the  laked  hemoglobin  may 
serve  to  produce  hemolysins,  but  they  obtained  only  questionable  results 
following  the  use  of  pure  hemoglobin.  Stewart  obtained  essentially 
the  same  results.  Nucleo-proteins  obtained  from  dog  blood  are  capable 
of  producing  specific  hemolysins.  Pearce  and  his  co-workers  have 
shown  that  nucleo-proteins  from  washed  organs  also  lead  to.  the  forma- 
tion of  hemolysins  specific  for  the  homologous  species.  Organ  and 
cell  extracts  free  from  blood  also  serve  as  hemolysinogens ;  the  best 
example  is  an  extract  of  spermatozoa,  for  in  this  instance  there  is 
no  question  of  blood  contamination  of  the  extract.  Of  further  interest 
is  the  fact  that  ether  extracts  of  erythrocytes,  alcohol-ether  extracts, 
and  extracts  in  1.5  per  cent,  sodium  bicarbonate  induce  the  formation 
of  weak  hemolysins  without  the  coincident  formation  of  hemagglu- 
tinins.  This  indicates  that  the  hemagglutinin  and  hemolytic  ambo- 
ceptor are  probably  separate  and  distinct  antibodies. 

Nature  of  the  Amboceptor. — The  amboceptor,  although  it  resists 
heat  of  56°  for  one  hour  or  more,  is  injured  by  heat  of  60°  C.  for  twenty 
minutes,  is  almost  completely  destroyed  by  70°  C.  for  one  hour  and  is 
completely  destroyed  by  boiling.  Like  antitoxin,  it  does  not  dialyze, 
is  electro-positive  and  is  resistant  to  ultra-violet  rays.  It  is  carried 
down  in  the  euglobulin  fraction  of  the  serum  protein,  but  by  various 
methods  of  purification  may  be  obtained  in  an  almost  protein-free 
state.  The  method  of  purification  described  by  Kosakai  is  of  im- 
portance from  various  points  of  view  and  deserves  some  description 
at  this  point.  He  requires  a  hemolytic  serum  which  titrates  I— 10,000. 
This  is  diluted  to  100  times  its  volume  with  salt  solution  and  5  c.c.  of 
the  diluted  serum  are  poured  into  4  c.c.  blood-cell  suspension.  The 
union  of  amboceptor  and  red  cells  is  accomplished  by  exposure  at  room 
temperature  for  fifteen  to  twenty  minutes,  after  which  the  cells  are 
freed  from  serum  by  repeated  washing.  To  the  antigen-amboceptor 
combination  is  added  isotonic  or  slightly  hypertonic  aqueous  solution 
of  a  sugar  such  as  saccharose,  glucose  or  lactose,  and  the  mixture  incu- 


CYTOLYSINS  125 

bated  at  55°  C.  for  fifteen  to  twenty  minutes,  during  which  period  it 
is  shaken  several  times.  The  mixture  is  centrif  uged  and  the  supernatant 
fluid  placed  in  a  separatory  funnel  with  five  to  ten  volumes  of  ether 
and  shaken  for  one  or  two  hours  until  the  solution  becomes  quite 
colorless.  The  saccharose  solution  is  separated  from  the  ether  and 
dialyzed  in  running  water  in  order  to  free  it  from  sugar  and  salt. 
After  dialyzation  the  solution  is  concentrated  in  a  vacuum  until  it 
reaches  the  original  volume  of  blood  serum  employed.  Strong  salt 
solution  may  prevent  amboceptor  from  entering  into  combination  with 
complement,  but  it  does  not  interfere  with  the  amboceptor  cell  union. 
Alkalis  may  prevent  either  form  of  union  and  may  serve  partly  to 
dissociate  amboceptor  cell  combinations. 

Mechanism  of  Operation  of  the  Amboceptor. — As  has  been 
pointed  out  previously,  the  action  of  amboceptor  is  differently  inter- 
preted by  the  Ehrlich  and  the  Bordet  schools.  If  the  Ehrlich  view  of 
the  two-fold  binding  group  is  to  be  adhered  to,  it  should  be  possible  to 
show  on  the  one  hand  a  combination  with  antigen,  and  on  the  other 
a  combination  with  complement.  Of  these  possibilities  there  is  no 
doubt  that  combination  with  cells  is  possible,  but  as  yet  no  conclusive 
evidence  has  been  produced  to  show  a  combination  between  comple- 
ment and  an  amboceptor  not  united  to  its  antigen.  The  discovery  of 
the  Neisser-Wechsberg  phenomenon  (see  page  147)  was  regarded  as 
demonstrating  a  combination  between  free  amboceptor  and  comple- 
ment. This  explanation,  however,  does  not  take  into  account  the 
possible  relationship  to  certain  colloidal  reactions  such  as  have  been 
described  in  connection  with  the  inhibition  zone  of  strong  agglutinins 
and  is  therefore  not  to  be  regarded  as  settled.  Ehrlich  and  Morgen- 
roth  stated  that  if  amboceptor  is  repeatedly  injected  into  animals  an 
anti-amboceptor  is  produced  which  serves  to  combine  with  the  cytophilic 
group  of  amboceptor,  but  Bordet  found  that  a  normal  serum,  free  from 
hemolytic  amboceptor  could  be  used  to  produce  the  same  immune  body, 
and  argued  therefrom  that  this  antibody  could  not  be  regarded  as  a 
specific  receptor.  Ehrlich  and  Sachs  admitted  the  fact  of  Bordet's 
experiments  and  came  to  the  conclusion  that  the  substance  is  anti- 
complementophile,  rather  than  anti-cytophile.  As  will  readily  be  seen 
this  argument  presupposes  the  correctness  of  the  Ehrlich  conception 
of  amboceptor,  and  is  therefore  not  to  be  accepted  as  conclusive.  With- 
out the  actual  demonstration  of  the  union  of  free  amboceptor  and 
complement,  the  union  of  antigenic  cells  and  amboceptor  is  of  quite  as 
much  value  in  support  of  the  Bordet  view  of  sensitization  as  in  support 
of  the  Ehrlich  hypothesis.  Nevertheless,  Ehrlich  and  Sachs  have 
reported  what  they  believe  to  be  a  crucial  experiment  in  that  it  appears 
to  show  that  at  least  in  some  instances  free  amboceptor  and  comple- 
ment may  combine.  Horse  serum  is  slightly  hemolytic  for  guinea-pig 
erythrocytes  and  ox  serum  is  somewhat  more  so.  If  inactivated  ox 
serum  and  fresh  horse  serum  are  added  to  guinea-pig  cells,  hemolysis 
occurs,  the  ox  serum  acting  presumably  as  an  amboceptor,  the  horse 
serum  as  complement.  If  the  guinea-pig  cells  are  treated  with  inac- 


126  THE  PRINCIPLES  OF  IMMUNOLOGY 

tivated  ox  serum  for  a  time  ordinarily  sufficient  for  amboceptor  ab- 
sorption, washed  free  of  serum  and  then  treated  with  fresh  horse 
serum  as  a  complement,  no  hemolysis  occurs.  Furthermore,  under 
these  conditions  no  hemolytic  immune  body  has  been  absorbed  from 
the  ox  serum.  Hemolysis  only  occurs  when  fresh  horse  serum  and 
inactivated  ox  serum  are  added  as  a  mixture.  The  interpretation  is 
that  in  this  particular  hemolytic  system  the  amboceptor  must  be  com- 
bined with  complement  before  the  amboceptor  combines  with  the  cells, 
or,  in  other  words,  that  the  complementophilic  group  of  a  free  ambo- 
ceptor has  united  with  complement  independently  of  the  cyto- 
philic  group. 

Conglutinin. — Bordet  and  Gay  have  studied  the  phenomenon  de- 
scribed in  the  preceding  paragraph  and  have  come  to  a  different 
conclusion  as  to  interpretation  because  of  their  discovery  of  a 
so-called  "  bovine  colloid  "  in  the  ox  serum.  They  attribute  the 
hemolysis  in  the  Ehrlich-Sachs  phenomenon  almost  entirely  to  the 
amboceptor  and  complement  of  the  horse  serum.  The  complex 
of  guinea-pig  cells  and  the  two  bodies  in  the  horse  serum  serves 
to  attract  the  bovine  colloid  which  augments  the  complementary  action 
of  the  horse  serum  so  as  to  produce  complete  hemolysis  and  at  the  same 
time  produces  marked  agglutination  of  the  cells.  This  colloid  is 
thermostable,  is  probably  of  protein  nature,  unites  with  a  complex  of 
cells,  amboceptor  and  complement,  but  does  not  act  upon  either  normal 
cells  or  cells  saturated  with  amboceptor.  Bordet  and  Streng  in  a  later 
study  named  the  colloid  "  conglutinin."  Streng  found  that  the  same 
phenonemon  could  be  demonstrated  in  regard  to  bacteriolysis  and  that 
conglutinin  is  present  in  the  sera  of  the  ox,  goat,  sheep  and  certain 
other  herbivora  but  not  in  the  sera  of  the  cat,  dog,  guinea-pig,  or  bird. 
Sachs  and  Bauer  have  not  offered  a  better  explanation  of  the  phe- 
nomenon unless  the  German  theory  of  amboceptor  is  unqualifiedly 
accepted.  In  our  opinion  both  sides  of  this  controversy  deserve  the 
most  careful  consideration  and  much  light  may  be  thrown  by  further 
study.  The  more  modern  views  of  immunological  processes,  influenced 
as  they  are  by  the  great  advances  in  colloidal  chemistry,  tend  toward 
acceptance  of  the  Bordet  hypothesis  of  sensitization  of  antigen  by  the 
thermostable  constituent  of  cytolytic  sera,  at  least  until  and  unless 
more  conclusive  contradictory  evidence  can  be  produced. 

Complement.  Distribution. — Complement  is  that  thermolabile  ele- 
ment of  normal  blood  which  in  the  presence  of  amboceptor  and  antigen 
completes  the  cytolytic  reaction.  As  regards  hemolysis,  complement 
in  the  presence  of  hemolytic  amboceptor  causes  solution  of  the  red 
blood-corpuscles  and  thus  renders  the  reaction  visible.  Complement 
is  found  in  the  blood  and  in  lesser  amount  in  nearly  all  the  other  body 
fluids  except  the  aqueous  humor  of  the  eye.  It  is  also  found  in  inflam- 
matory exudates  and  sometimes  in  transudates,  but  it  is  not  present 
in  the  urine,  nasal  secretion  or  the  secretion  of  other  glands  except 
that  of  the  breast  (milk).  The  amount  in  the  blood  is  fairly  constant 
for  any  given  individual,  but  during  the  first  twenty-four  hours  after 


CYTOLYSINS  127 

birth  the  complement  content  of  the  blood  has  been  found  to  be  rather 
small;  Gay  has  found  it  to  be  somewhat  less  in  women  than  in  men. 
Moro  has  found  it  to  be  less  in  bottle-fed  than  breast-fed  babies. 
Although  individual  variation  may  be  great,  there  is  a  certain  uni- 
formity in  different  members  of  the  same  species.  This  is  true  through- 
out a  large  number  of  species,  except  the  horse,  in  which  species  it  is 
found  to  vary  markedly.  Different  species  as  such  contain  different 
amounts  of  complement.  The  guinea-pig  contains,  as  a  rule,  more 
complement  per  cubic  centimeter  than  other  species.  Man  and  rabbit 
contain  less  than  the  guinea-pig,  and  in  the  case  of  the  mouse  it  is 
very  difficult  to  demonstrate  any  complement  at  all.  It  has  recently 
been  found  that  insects  and  mollusks  contain  practically  no  complement. 
Alterations  of  Amount  of  Complement. — The  amount  of  comple- 
ment in  a  given  blood  may  be  made  to  vary  by  artificial  means.  For 
example,  the  injection  of  indifferent  materials,  such  as  foreign  blood 
plasma,  bouillon,  aleuronat,  pepton,  yeast,  nuclein,  physiological  salt 
solution,  produces  an  increase  in  the  amount  of  complement,  but  this 
increase  is  not  permanent.  Similarly  complement  may  be  increased  for 
a  short  time  following  the  injection  of  pilocarpin,  phlorizin,  staphylo- 
cocci,  oil  of  turpentine  and  thyreoidin ;  exposing  an  animal  to  high 
temperatures  may  also  increase  complement.  Although  it  is  generally 
true  that  complement  is  not  increased  by  immunization,  nevertheless 
Cantacuzene  has  recently  shown  that  by  injecting  red  blood-corpuscles 
into  certain  marine  invertebrates  he  is  able  to  increase  the  amount  of 
complement  in  their  blood.  Complement  may  be  reduced  temporarily 
by  the  injection  of  sodium  taurocholate,  potassium  picrate,  toluylendi- 
amin  and  more  permanently  by  experimental  phosphorus  poisoning, 
experimental  chronic  suppuration,  starvation  and  by  alcohol  poisoning. 
If  sensitized  blood-cells,  i.e.,  blood-cells  saturated  with  amboceptor, 
are  injected  into  an  animal,  it  can  be  demonstrated  that  the  amount  of 
complement  is  reduced  by  the  hemolysis  which  takes  place  in  vivo. 
Shaw  has  found  that  in  the  case  of  recently  acquired  syphilis,  although 
the  blood  before  treatment  shows  no  alteration  of  complementary  ac- 
tivity, yet  the  administration  of  salvarsan  may  reduce  this  activity 
to  a  considerable  degree.  The  experimental  investigations  of  the  effect 
of  disease  in  man  on  the  complement  content  of  his  blood  are  very 
unsatisfactory  because  human  blood  normally  contains  only  a  small 
amount  of  complement  and  the  detection  of  any  variation  is  susceptible 
to  a  wide  margin  of  experimental  error. 

Method  of  Obtaining  Complement. — Complement  is  usually  obtained 
from  the  guinea-pig-,  although  under  special  circumstances  it  may  be  obtained 
from  other  animals.  The  blood  may  be  withdrawn  in  any  manner  adapted  to 
such  a  procedure.  In  the  case  of  the  guinea-pig  the  method  employed  in  this 
laboratory  is  to  anesthetize  the  animal  very  slightly,  pull  the  hair  from  the 
neck,  make  a  longitudinal  slit  in  the  mid-line  of  the  neck,  place  a  15  c.c.  centri- 
fuge tube  toward  the  upper  end  of  the  slit  with  its  lip  firmly  pressed  into  the 
opening,  then  with  a  scissors  snip  the  carotid  artery,  carefully  avoiding  the 
trachea.  The  animal  is  then  held  head  downward  while  the  blood  drains  into 
the  tube.  The  blood  is  allowed  to  clot  in  the  tube  and  the  clot  separated  from 
the  side  of  the  tube  with  a  long  sterile  or  clean  needle,  as  the  necessity  of  the 
case  indicates.  The  clot  separates  best  at  room  temperature,  but  if  centrifuga- 


128 


THE  PRINCIPLES  OF  IMMUNOLOGY 


tion  cannot  be  done  immediately  the  clot  may  be  allowed  to  separate  in  the 
ice  chest.  As  soon  as  the  serum  has  separated  out  of  the  clot,  the  tube  is 
centrifuged  and  the  serum  collected  by  means  of  a  pipette.  If  guinea-pigs  are 
large,  the  blood  may  be  collected  in  smaller  quantities  by  heart  puncture  or  by 
bleeding  from  an  ear  vein,  thus  obviating  the  necessity  for  killing  the  animal. 


FlG.  14. — Method  of  obtaining  blood  from  guinea-pig  (See  text). 


Origin  of  Complement. — Considerable  controversy  has  been  waged 
concerning  the  origin  of  complement  since  the  time  that  Hankin  and 


CYTOLYSINS  129 

subsequently  Metchnikoff  expressed  the  belief  that  complement  origin- 
ates in  the  leucocytes  of  the  body  and  is  only  liberated  upon  the»death 
of  these  cells.  Metchnikoff  used  the  term  cytase  to  indicate  what  we 
now  call  complement  and  believed  that  the  microphages  gave  rise  to 
a  microcytase  capable  of  dissolving  blood  and  other  body  cells.  Pfeiffer 
and  certain  other  German  workers  take  a  diametrically  opposed  posi- 
tion and  maintain  that  the  leucocytes  furnish  none  of  the  complement  in 
the  blood.  A.  von  Wassermann  and  also  Landsteiner  believe  that  the 
leucocytes  may  constitute  one  source  of  origin  for  the  complement, 
and  it  seems  practically  certain  from  modern  investigations  that  several 
organs  play  a  part  in  the  formation  of  complement.  Before  the  bac- 
tericidal action  of  blood  was  thoroughly  understood  as  due  to  the  inter- 
action of  amboceptor  and  complement,  certain  studies  seemed  to  indicate 
that  exudates  rich  in  leucocytes  were  active  as  bactericidal  agents,  but 
it  is  now  understood  that  other  constituents  of  the  exudate  take  part 
in  this  phenomenon  and  more  recent  experiments  show  that  extracts 
of  leucocytes  do  not  yield  a  complement.  It  has  been  shown  further 
that  variations  in  the  total  leucocyte  count  in  an  animal  produce  no 
corresponding  variations  of  complement  content.  Neuf  eld  and  certain 
others  take  the  view  that  even  inside  the  living  leucocytes  there  is  no 
complement  because  they  have  found  that  destruction  of  red  blood- 
corpuscles  within  living  leucocytes  takes  place  at  a  distinctly  slower 
rate  of  speed  than  is  the  case  in  ordinary  hemolysis.  Furthermore, 
they  point  out  that  the  method  of  destruction  is  quite  different,  in  that 
ordinary  hemolysis  shows  simply  liberation  of  hemoglobin  without 
destruction  of  the  stroma.  Metchnikoff 's  belief  that  the  death -of  the 
leucocytes  yields  complement  was  supported  by  an  experiment  which 
apparently  showed  that  complement  is  present  in  serum  after  clotting, 
but  not  in  plasma.  A  considerable  amount  of  experimental  evidence 
has  been  adduced,  since  this  statement  of  Metchnikoff,  to  show  that 
plasma  contains  complement  in  the  same  amount  as  does  serum.  Some 
of  these  experiments  appeared  to  be  invalid  on  the  ground  that  im- 
munological  work  with  a  plasma  is  likely  to  lead  to  coagulation,  thus 
producing  a  serum  for  the  actual  experiments.  After  these  objections 
had  been  presented,  further  experiments  were  performed  which  over- 
came such  objection,  and  it  now  seems  perfectly  clear  that  plasma  con- 
tains complement.  This  fact  has  been  firmly  established  by  the 
recent  work  of  Watanabe. 

Nature  of  Complement. — Complement  is  probably  of  protein 
nature,  inasmuch  as  it  is  destroyed  in  coagulation  of  the  serum  by  heat 
and  is  digested  by  trypsin.  Noguchi  and  his  co-workers  were  of  the 
opinion  that  complement  is  a  combination  of  soap  and  a  protein,  but 
numerous  other  workers  failed  to  confirm  these  studies.  This  state- 
ment of  Noguchi,  as  well  as  the  work  of  Kyes,  with  cobra  venom  led 
to  the  hope  that  it  might  be  possible  to  prepare  an  artificial  complement. 
Landsteiner  and  Jagic  have  investigated  the  question  and  have  shown 
that  whereas  it  is  possible  to  substitute  for  amboceptor  a  colloidal 
solution  of  silicic  acid,  which  nevertheless  shows  none  of  the  specific 
9 


130  THE  PRINCIPLES  OF  IMMUNOLOGY 

characters  of  amboceptor,  it  is  absolutely  impossible  up  to  the  present 
time  to  offer  any  substitute  for  complement.  Complement  resembles 
an  enzyme  in  that  it  is  thermolabile,  disintegrates  cells,  does  not  pass 
through  Berkefeld  filters,  is  adsorbed  by  kaolin  and  destroyed  by 
shaking.  Furthermore,  it  activates  amboceptor  much  in  the  same 
manner  as  entero-kinase  activates  trypsinogen.  As  against  the  idea  that 
complement  is  an  enzyme  is  the  fact  that  in  the  reaction  of  hemolysis, 
hemoglobin  is  liberated  without  destruction  of  the  stroma  of  the  cells 
and  the  further  fact  that  complement  acts  quantitatively,  following  in 
a  general  way  the  law  of  multiple  proportions.  As  is  well  known,  heat 
at  56°  to  60°  C.  for  one-half  hour  destroys  the  complementary  activity 
of  a  serum.  It  has  recently  been  shown,  however,  that  if  heat  of 
56°  C.  is  applied  for  only  a  short  period,  i.e.,  from  seven  to  te.n  minutes, 
the  complementary  action  is  restored  after  several  hours  have  elapsed 
(the  phenomenon  of  Gramenitski).  This  is  interpreted  as  due  to  an 
agglomeration  or  aggregation  of  protein  particles  resembling  heat 
coagulation  of  protein.  The  restoration  of  activity  after  standing  is 
ascribed  to  a  dispersion  of  the  protein  aggregates  so  that  they  can 
act  nearly  or  quite  as  they  did  originally.  Ultra-violet  rays  destroy 
complement,  but  it  is  stated  that  X-rays  do  not.  Recent  work  in  this 
laboratory  by  Ecker  has  shown  that  the  visible  spectrum  also  serves  to 
reduce  complementary  activity.  Experimental  conditions  in  this  in- 
stance made  it  possible  to  work  with  three  divisions  of  the  spectrum, 
namely,  a  division  near  the  violet  end,  a  division  in  the  middle  of  the 
spectrum  and  a  division  near  the  red  end.  It  was  found  that  those  rays 
toward  the  violet  end  of  the  spectrum  were  more  active  than  the 
rays  in  the  middle  of  the  spectrum  and  the  latter  were  more  active 
than  the  rays  at  the  red  end  of  the  spectrum.  That  this  is  a  function  of 
the  wave-length  of  the  ray  is  not  absolutely  certain  but  seems  probable 
in  view  of  the  work  of  Bovie,  Brooks  and  others,  which  shows  that  the 
presence  of  cells  in  the  serum  reduces  the  activity  of  the  ultra-violet 
rays.  That  the  destruction,  however,  is  a  function  of  the  penetrability 
of  the  rays  is  not  borne  out  by  the  statement  that  X-rays  fail  to 
destroy  complement.  We  have  also  been  able  to  show  in  this  lab- 
oratory that  drying  of  complement  produces  some  deterioration.  Other 
workers  have  stated,  however,  that  if  the  complement  is  mixed  with 
a  proper  concentration  of  salt,  preferably  about  8  per  cent,  -and  then 
dried,  the  salting  nullifies  the  destructive  action  of  desiccation  and 
the  dried  serum  under  these  circumstances  may  be  preserved  for  a 
considerable  period  of  time.  Complement  may  be  inhibited  by  the 
presence  of  hydroxyl  ions  but  is  restored  to  activity  by  the  addition  of 
hydrogen  ions.  Complement  can  be  made  to  combine  with  magnesium, 
calcium,  barium,  strontium  and  sulphate  ions  and  can  be  separated  by 
simple  chemical  precipitation.  Acids  and  alkalis  in  sufficient  concen- 
tration also  serve  to  destroy  complement. 

Preservation  of  Complement. —  Owing  to  the  extreme  lability  of 
complement,  the  question  of  prolonged  preservation  assumes  consid- 
erable importance.  The  fresh  serum  may  be  desiccated  in  air,  in 


CYTOLYSINS  131 

vacuum,  in  vacuum  after  freezing,  or  on  filter  paper.  In  the  hands  of 
certain  workers  various  methods  of  this  sort  have  proven  more  or 
less  successful  but  do  not  seem  to  be  widely  applicable.  It  is  of  im- 
portance to  keep  in  mind  that  under  such  conditions  the  desiccation 
of  serum  does  not  remove  the  possibility  of  the  destructive  action  of 
light.  Other  methods  of  preservation  include  salting  with  sodium 
chloride  and  also  with  sodium  acetate.  The  former  has  been  fairly 
successful,  but  the  latter  has  been  completely  abandoned.  Another 
method  is  salting  and  then  freezing,  but  this  has  been  found  to  be  in 
no  way  superior  to  freezing  without  salting.  According  to  Bigger, 
it  is  of  extreme  importance  that  the  serum  should  be  sterile  to  ensure 
the  success  of  any  method  of  preservation.  Browning  and  Mackie  have 
found  that  frozen  serum  kept  at  a  temperature  of  —15°  C.  retains  its 
complementary  power  three  months  without  appreciable  loss.  Noguchi 
and  Bronfenbrenner  found  that  at  10°  C.  the  serum  loses  one-half 
its  original  strength  at  the  end  of  twenty-four  hours.  If  it  is  kept  at 
37°  C.  it  loses  two-fifths  of  its  strength  at  the  end  of  six  hours;  at 
45°  C.  one-half  hour  exposure  reduces  it  to  one-third  to  one-half  its 
original  strength ;  at  50°  C.  50  per  cent,  is  lost  in  five  minutes.  They 
have  examined  the  rate  of  destruction  at  55°  and  find  that  this  goes 
on  quite  irregularly  and  is  not  in  proportion  to  the  length  of  time. 
The  irregularity,  however,  presents  a  certain  rhythm,  i.e.,  a  period  of 
greater  destruction  alternating  with  a  period  of  less  active  destruction. 
Reudiger  has  studied  the  preservation  of  frozen  complement  and  finds 
that  at  the  expiration  of  one  week  whether  the  complement  is  made 
up  of  serum  of  a  single  guinea-pig  or  the  pooled  serum  of  several 
guinea-pigs  the  activity  in  the  Wassermann  is  somewhat  stronger  than 
with  fresh  serum.  At  the  end  of  two  weeks  the  frozen  complement 
gives  results  that  are  practically  identical  with  the  results  obtained 
with  fresh  complement,  but  after  two  weeks  the  frozen  complement 
gradually  loses  strength  apparently  more  rapidly  in  mild  weather  than 
in  very  cold  weather. 

Variability  of  Complement. — Complementary  activity  varies  con- 
siderably in  different  sera ;  in  the  same  serum  it  may  operate  differently 
with  amboceptors  from  several  different  species.  The  explanation  of 
this  difference  of  activity  has  led  to  a  difference  of  opinion  as  to  the 
exact  nature  of  the  complementary  activity.  Ehrlich  and  Morgenroth 
and  the  German  school  take  the  position  that  a  given  serum  contains  a 
considerable  number  of  complements,  whereas  Bordet  and  his  school 
take  the  point  of  view  that  the  complement  in  any  given  serum  is  a  unit, 
although  they  admit  that  complements  in  different  sera  may  represent 
a  somewhat  different  constitution. 

Multiplicity  of  Complements. — Ehrlich  and  Morgenroth  were  able 
to  show  that  the  complementary  activity  of  a  serum  could  be  divided 
by  means  of  filtration  in  the  following  respect.  They  showed  that 
complement  for  sensitized  guinea-pig  cells  passes  through  the  filter, 
whereas  complement  for  sensitized  rabbit  cells  remains  in  the  filter. 
It  has  also  been  shown  by  thermal  and  chemical  differentiation  that 


132  THE  PRINCIPLES  OF  IMMUNOLOGY 

some  complements  are  destroyed  and  others  remain  in  the  same  serum/ 
Weak  acids  and  weak  alkalis  may  differentiate  complement  similarly; 
it  is  stated  that  digestion  by  papain  also  serves  so  to  differentiate.  By 
injection  of  a  complementary  serum  into  foreign  species,  a  so-called 
anti-complement  is  obtained  which  is  said  to  act  upon  one  of  the 
complements  of  a  serum  and  not  upon  others,  irrespective  of  whether 
the  complement  of  the  antigenic  serum  or  of  some  other  serum  be  em- 
ployed in  the  subsequent  test.  Practically  all  these  experiments  have 
been  performed  in  such  a  way  that  the  complement  acts  with  normal 
amboceptors  and  the  question  at  once  arises  as  to  whether  or  not  the 
same  phenomena  would  be  observed  with  immune  amboceptors.  Even 
in  the  case  of  normal  amboceptors,  there  is  experimental  contradiction 
of  the  original  supposition  of  Ehrlich  and  Morgenroth.  Neisser  stated 
that  anthrax  bacilli  deprive  fresh  rabbit  serum  of  its  bactericidal  com- 
plement, but  not  of  its  hemolytic  complement.  Wilde  showed  that  if 
a  sufficient  mass  of  anthrax  bacilli  were  added  to  the  fresh  rabbit  serum 
all  the  complement  is  used,  so  that  further  bactericidal  action  does  not 
occur  and  no  hemolytic  action  can  be  demonstrated.  Similarly  Bordet 
found  that  unsensitized  red  blood-corpuscles  deprive  a  serum  of  only 
part  of  its  complement  but  that  cells  strongly  sensitized  with  hemolysin 
use  up  all  the  complement  both  bactericidal  and  hemolytic.  He  believes 
that  the  reason  normal  amboceptors  do  not  utilize  all  the  available 
complement  is  due  to  the  fact  that  such  normal  amboceptors  do  not  suf- 
ficiently sensitize  the  antigenic  cells.  Therefore,  the  complete  sensitiza- 
tion  of  the  cells  will  result  in  a  complete  utilization  of  complement. 
After  the  publication  of  these  experiments,  Ehrlich,  who  confirmed  the 
results,  explained  the  phenomenon  as  being  due  to  a  multiplicity  of 
complements  in  the  serum.  In  order  to  do  so,  he  was  obliged  to  alter 
the  original  conception  of  the  amboceptor,  so  that  instead  of  having 
a  single  cytophilic  group  it  must  contain  several  cytophilic  groups. 
Therefore,  the  term  was  altered  to  polyceptor  instead  of  amboceptor. 
The  polyceptor  was  supposed  to  have  one  group  with  an  especial  affinity 
for  a  dominant  complement  and  other  receptors  with  affinities  for  the 
secondary  complements.  If  the  dominant  complement  is  absorbed  by 
the  polyceptor,  the  secondary  complements  are  also  involved,  but,  on 
the  other  hand,  if,  as  has  been  claimed,  it  is  possible  to  obtain  a  serum 
with  only  secondary  complements  present,  these  may  be  absorbed 
without  action  upon  the  receptor  for  dominant  complement.  This  expla- 
nation, however,  rests  entirely  upon  the  Ehrlich  conception  of  the  ambo- 
ceptor, and,  inasmuch  as  this  conception  is  not  conclusively  proven,  it 
is  not  necessary  to  accept  the  idea  that  complements  are  multiple.  This 
question,  however,  is  not  settled  at  the  present  time,  and  reference 
will  be  made  to  it  again  in  connection  with  the  phenomenon  of  com- 
plement fixation. 

Complementoids. — The  similarity  in  action  and  nature  of  comple- 
ment and  toxin  was  early  recognized,  and  it  was  therefore  attempted  to 
determine  whether  or  not  complement  could  be  broken  up  in  the  same 
way  as  toxin  so  as  to  form  complementoids.  If  such  were  the  case,  it 


CYTOLYSINS  133 

should  be  possible  to  break  up  a  complement  so  as  to  demonstrate  a 
haptophore  group  and  a  zymophore  or  zymotoxic  group.  Thus,  ex- 
posure to  increased  temperature  might  be  so  arranged  as  to  destroy 
the  zymophore  group  and  leave  the  haptophore  group  intact.  If  this 
were  true,  the  haptophore  group  or  complementoid  might  be  added  to 
an  antigen-amboceptor  mixture  and  thus  prevent  any  further  action 
by  a  fresh  whole  complement.  Experimentally,  however,  it  was  found 
that  this,  in  the  majority  of  instances,  does'  not  occur.  Nevertheless, 
Ehrlich  and  Sachs  found  that  if  they  mixed  inactivated  guinea-pig 
serum,  normal  inactivated  dog  serum,  and  guinea-pig  cells,  hemolysis 
did  not  occur,  even  after  the  addition  of  fresh  guinea-pig  serum.  They 
believed  this  to  be  the  result  of  a  blocking  or  plugging  of  the  com- 
plementophile  group  of  the  dog  amboceptor  by  the  complementoid  of 
the  inactivated  guinea-pig  serum,  thereby  preventing  the  union  when 
fresh  active  complement  was  added.  Fuhrmann  supported  this  state- 
ment and  maintained  that  allowing  the  complement  to  stand  for  a 
period  of  three  weeks  was  even  more  adapted  to  separation  of  the  hapto- 
phore and  zymophore  group.  Following  this  work,  Muir  and  Brown- 
ing conducted  an  extensive  series  of  complicated  experiments,  which 
in  the  main  tend  to  support  the  view  of  Ehrlich  that  complementoids 
actually  exist.  If  they  do  exist,  however,  they  are  not  uniformly  demon- 
strable, and  it  may  very  well  be  that  this  is  due  to  the  difference  in 
destructibility  of  the  two  groups  being  so  slight  that  our  methods  of 
differentiating  by  means  of  heat  and  standing  are  not  sufficiently  exact. 
Complement  Fractions. — Further  light  was  thrown  on  the  possi- 
bility of  fractioning  complement  by  the  experiments  of  Ferrata,  who 
found  that  dialyzation  of  the  serum  resulted  in  the  destruction  of  com- 
plementary activity.  Dialyzation  precipitates  the  so-called  globulin 
fraction  of  the  serum  as  contrasted  with  the  albumin  fraction  which 
remains  in  solution.  The  precipitate  may  be  dissolved  in  physiological 
saline  and  the  portion  in  solution  may  be  restored  to  its  original  salt 
concentration,  thereby  forming  isotonic  solutions  of  the  two  protein  frac- 
tions. Ferrata  found  that  neither  of  these  components  in  the  presence  of 
an  amboceptor  was  capable  of  producing  hemolysis,  but  that  if  both  were 
added,  sufficiently  soon  after  dialyzation,  hemolysis  would  take  place. 
Brand  studied  this  phenomenon  further  and  found  that  both  fractions 
are  equally  thermolabile  and  because  of  activities  which  he  discovered, 
named  the  fraction  contained  in  the  globulin  precipitate  "  mid-piece  "  and 
that  in  the  albumin  "  end-piece."  If  the  amboceptor- cell  mixture  is 
treated  first  with  mid-piece,  no  hemolysis  occurs,  but  if  end-piece  is  then 
added,  hemolysis  occurs  as  it  would  have  in  the  original  amount  of  com- 
plement. If  the  end-piece  is  first  added  and  later  the  mid-piece,  hemo- 
lysis will  occur,  but  in  very  much  smaller  degree  than  if  the  entire 
complement  had  been  used.  Zinsser  found,  however,  that  when  mid- 
piece  and  end-piece  are  mixed  and  then  added  to  the  sensitized  cells, 
the  hemolytic  effect  is  reduced  and  is  considerably  less  than  if  mid-piece 
is  added  before  end-piece.  It  has  been  found  that  the  mid-piece  may 
enter  into  combination  with  the  sensitized  cells  at  o°  C,  but  the  end- 


I34  THE  PRINCIPLES  OF  IMMUNOLOGY 

piece  combines  only  at  considerably  higher  temperatures.  It  has  also 
been  found  that  the  mid-piece  of  one  animal  species  may  be  activated 
by  the  end-piece  of  another  animal  of  the  same  or  different  species. 
Nevertheless,  Ritz  and  Sachs  have  shown  that  the  serum  of  an  animal 
may  possess  a  mid-piece  for  certain  sensitized  erythrocytes,  but  does 
not  necessarily  possess  a  corresponding  end-piece.  Marks  has  studied 
the  quantitative  relations  of  mid-piece  and  end-piece  and  has  found  that 
a  ratio  of  i-i  is  not  necessarily  the  optimum  for  hemolysis  and  that 
very  often  it  is  necessary  to  change  the  ratio;  this  change  must  be  by 
increase  of  mid-piece,  never  by  increase  of  end-piece.  If  the  two 
are  mixed  before  addition  to  the  amboceptor-cell  mixture,  an  excess 
of  mid-piece  inhibits  hemolysis,  but  if  the  excess  of  mid-piece  is  added 
first  followed  by  end-piece,  hemolysis  is  complete.  Brand  and  later 
Hecker  found  that  if  the  globulin  precipitate  is  preserved  dry  or  in 
solution  in  distilled  water  it  will  retain  activity  for  several  days,  but  in 
physiological  salt  solution  it  loses  its  activity  in  three  to  four  hours. 
This,  however,  does  not  indicate  destruction  of  mid-piece  in  salt  solu- 
tion since  it  will  combine  with  sensitized  cells  if  added  before  end- 
piece.  Marks  holds  that  this  phenomenon  is  due  to  the  inhibition  of 
hemolysis  by  excess  of  mid-piece  and  does  not  occur  if  proper  pro- 
portions are  maintained  in  the  mixture.  Swirski  maintains  that  the 
complement  fixation  of  the  Wassermann  test  binds  mid-piece  but  not 
end-piece.  This  has  been  investigated  by  Bronf  enbrenner  and  Noguchi, 
who  believe  that  the  free  end-piece  in  Wassermann  tests  differs  from  all 
other  end-pieces  in  that  it  activates  the  complex  which  includes  sheep 
cells  but  has  no  effect  upon  the  cells  of  other  animals.  Bessemans  has 
recently  investigated  again  the  question  of  thermostability  of  end-piece 
and  mid-piece.  He  finds  that  there  are  important  differences  in  certain 
of  the  sera  he  has  examined,  so  that  a  general  statement  in  regard  to  the 
heat  resistance  of  these  fractions  is  not  justified. 

Browning  and  Mackie  have  found  that  by  various  methods  of  f rac- 
tioning  the  serum  it  is  possible  to  divide  complement  into  four  frac- 
tions and  that  certain  combinations  consisting,  as  a  rule,  of  at  least  three 
of  these  reproduce  quantitatively  the  full  hemolytic  effect  of  the  whole 
complement.  This  presents  numerous  intricate  possibilities  of  experi- 
ment, but  the  important  point  is  that  such  a  demonstration  makes  the 
use  of  the  terms  mid-piece  and  end-piece  no  longer  desirable. 

Normal  Hetero-hemolysins. — The  preceding  discussion  has  been 
concerned  chiefly  with  complement  and  immune  amboceptor.  Histori- 
cally much  study  had  been  directed  toward  the  normal  cytotoxic  powers 
of  blood  serum  before  the  immune  amboceptors  were  recognized; 
Fodor,  Nuttall,  Nissen  and  Buchner  had  investigated  the  action  of  nor- 
mal sera  in  dissolving  bacteria.  Buchner  in  1893  pointed  out  a  similar 
capacity  of  blood  serum  for  dissolving  animal  cells,  particularly  ery- 
throcytes. Ehrlich  and  Morgenroth  took  up  the  question  as  to  whether 
or  not  the  globulicidal  activity  of  normal  serum  is  due  to  an  interaction 
of  two  substances  similar  to  that  in  immune  sera.  They  showed  that 
normal  dog  serum  is  capable  of  dissolving  guinea-pig  erythrocytes,  but 


CYTOLYSINS  135 

that  it  is  inactivated  by  heating  to  55°  C.  Reactivation  by  fresh  dog 
serum  was  undesirable  because  of  the  normal  amboceptor  present. 
Therefore,  they  employed  fresh  guinea-pig  serum  in  fairly  large  doses 
and  reactivated  the  heated  dog  serum  so  that  complete  hemolysis  oc- 
curred. Thus  they  demonstrated  the  double  nature  of  the  normal 
hemolysins  and  also  that  a  complement  may  serve  to  hemolyze  cells 
of  the  same  species  from  which  the  complement  is  obtained.  Other 
experiments  have  shown,  however,  that  a  complement  operates  less 
actively  against  homologous  cells  than  against  heterologous  cells.  Ehr- 
lich  and  Morgenroth  showed  similar  relationships  by  employing  as  the 
amboceptor  normal  calf  serum  and  normal  sheep  serum,  as  well  as 
similar  hemolytic  complexes  with  sheep  and  goat  blood.  They  also 
showed  that  in  a  number  of  instances .  the  amboceptor  could  be  dif- 
ferentially absorbed  by  cells  at  o°  C.,  leaving  complement  free  in  the 
serum.  Such  absorption  could  not  be  accomplished  with  all  normal 
hemolytic  sera;  in  some  the  cells  absorbed  both  amboceptor  and  com- 
plement, whereas  in  others  no  absorption  whatever  occurred.  They 
interpreted  the  absorption  of  both  bodies  as  due  to  the  possession  on  the 
part  of  the  amboceptor  of  equal  avidity  of  both  the  cytophile  and  com- 
plementophile  group,  whereas  failure  of  absorption  was  supposed  to  be 
due  to  a  stronger  affinity  of  complement  for  amboceptor  than  of  cells 
for  amboceptor.  We  record  the  fact  without  accepting  the  explanation, 
but  it  is  important  that  in  some  instances  normal  hemolytic  sera  may 
fail  to  exhibit  a  separability  of  complement  and  amboceptor  by  means 
of  differential  absorption. 

A  normal  serum  may  be  hemolytic  for  cells  of  more  than  one  species  ; 
this  is  true  of  goat  serum,  which  is  hemolytic  for  both  guinea-pig  and 
rabbit  cells.  In  such  cases  it  is  possible  to  absorb  one  amboceptor,  leave 
the  other  active  in  the  serum  and  thus  demonstrate  multiplicity  of 
specific  amboceptors  in  a  serum.  Ehrlich  and  Morgenroth  also  main- 
tained that  in  the  case  of  goat  blood  there  is  a  different  complement  for 
the  two  types  of  cells,  but  as  has  been  indicated  in  discussing  comple- 
ment this  possibility  seems  unlikely. 

Proportions  of  Amboceptor  and  Complement  in  Normal 
Hemolysins. — Further  difference  between  a  normal  and  immune 
hemolytic  serum  lies  in  the  different  proportion  of  amboceptor  and 
complement.  In  a  normal  hemolytic  serum  the  amount  of  amboceptor 
present  is  small  and  the  complement  is  usually  present  in  at  least  suf- 
ficient quantity  to  saturate  the  amboceptor ;  it  may  be  present  in  excess. 
In  immune  sera  the  amboceptor  is  increased  enormously,  whereas  the 
complement  is  not  altered  in  quantity.  Therefore,  such  an  immune 
serum  may  contain  amboceptor  in  greater  quantities  than  can  be  sat- 
urated by  complement  and  for  its  full  activity  requires  more  com- 
plement than  can  be  furnished  in  the  immune  animal's  serum.  This 
increase  in  amboceptor  is  out  of  all  proportion  to  the  amount  of  antigenic 
cells  injected.  Muir,  for  example,  calculated  that  in  immunizing  a 
rabbit  the  total  amount  of  ox  blood  injected  was  30  c.c.,  and  hemolytic 
amboceptor  was  produced  sufficient  to  dissolve  the  erythrocytes  in 


136  THE  PRINCIPLES  OF  IMMUNOLOGY 

6000  c.c.  of  ox  blood.  The  practical  bearing  of  the  disproportion  of  com- 
plement to  immune  amboceptor  lies  in  the  use  of  bactericidal  sera.  It 
is  easily  conceivable  that  injections  of  such  sera  may  meet  in  the  injected 
animals'  blood  with  an  insufficient  amount  of  complement  for  complete 
activation.  Therefore,  it  may  be  advisable  in  such  experiments  or  in 
therapeutic  use  of  sera  of  this  type  to  activate  with  a  sufficient  quantity 
of  fresh  complementary  serum. 

Normal  Iso-hemolysins. — Attention  has  been  called  (page  99)  to 
the  phenomenon  of  iso-hemagglutination.  Similarly  iso-hemolysins 
can  be  demonstrated.  For  such  a  purpose  the  serum  must  be  fresh 
and  the  corpuscles  exposed  to  it  at  incubator  temperature.  It  is  prob- 
able that  these  hemolysins  resemble  other  normal  hemolysins.  The 
groups  correspond  to  the  groups  of  iso-hemagglutinins.  As  in  other 
experiments  with  hemolysins,  agglutination  appears  and  is  followed 
by  hemolysis.  Agglutination  inhibits  hemolysis  to  a  certain  degree,  as 
has  been  shown  by  Handel  and  by  Karsner  and  Pearce.  Kolmer,  Trist 
and  Flick  have  maintained  in  a  recent  study  that  there  are  two  varieties 
of  natural  hemolysin  and  hemagglutinin  in  human  sera.  They  find  a 
thermolabile  variety  of  these  antibodies  which  is  destroyed  at  56°  C. 
for  thirty  minutes  and  a  less  thermolabile  or  thermostable  body  which 
is  destroyed  at  62°  C.  for  thirty  minutes.  Exposure  at  56°  C.  removes 
the  various  iso-hemolysins  but  does  not  destroy  the  iso-hemagglutinins. 
Sands  and  West  have  found  that  if  the  immune  sera  are  filtered  (more 
particularly  in  I— 10  dilution)  through  perfectly  clean  Kitasato  or  Cham- 
berland  filters  a  large  amount  of  the  hemagglutinin  is  removed,  with 
slight  or  no  reduction  of  hemolytic  activity.  In  fact,  the  hemolytic 
activity  may  be  increased  by  the  filtration  and  this  may  be  explained 
as  due  to  the  removal  of  whatever  inhibiting  power  on  hemolysis 
hemagglutination  may  display. 

Anti-amboceptors. — Inasmuch  as  the  bodies  which  take  part  in 
hemolysis,  the  amboceptor  and  complement,  are  of  protein  nature,  it 
is  presumable  that  they  might  serve  as  antigenic  substances.  It  should 
be  possible  to  prepare  anti-amboceptor  and  anti-complement.  As  pre- 
viously mentioned,  experiments  have  been  reported  which  have  been 
interpreted  to  indicate  that  it  is  possible  to  produce  such  immune  anti- 
complements,  but  the  evidence  offered  has  not  withstood  criticism ;  at 
the  present  time  it  is  extremely  unlikely  that  true  anti-complements 
have  been  demonstrated.  Anti-amboceptors  were  first  produced  by  Ehr- 
lich  and  Morgenroth  who  injected  as  the  antigen  a  hemolytic  immune 
serum.  If  a  hemolytic  immune  serum  is  injected  in  fairly  large  amounts 
into  an  animal  for  whose  red  cells  the  serum  is  specific,  death  results. 
By  carefully-graded  injections,  however,  it  is  possible  so  to  immunize 
the  animal  that  it  becomes  immune  to  the  toxic  effect  of  the  serum. 
When  so  immunized  the  serum  of  this  animal  when  added  to  a  cell 
amboceptor  mixture  and  incubated  will  prevent  subsequent  hemolysis 
on  the  addition  of  complement.  Similarly  anti -amboceptor s  may  be 
produced  by  injecting  serum  containing  amboceptors  into  other  animals 
than  those  for  which  the  serum  is  hemolytic.  Ehrlich  and  Morgenroth 


CYTOLYSINS  137 

were  of  the  opinion  that  such  anti-amboceptors  represented  an  excess 
of  cell  receptors  formed  during  the  course  of  immunization  and  were 
thus  free  in  the  serum  to  combine  with  the  cytophilic  group  of  the 
amboceptor.  Bordet  found,  however,  that  it  was  not  necessary  to  use 
a  hemolytic  immune  serum  as  an  antigen  and  demonstrated  an  anti- 
amboceptor  by  injecting  normal  rabbit  serum  into  guinea-pigs.  The 
anti-serum  formed  in  the  guinea-pig  not  only  neutralized  hemolytic 
amboceptor  of  rabbit  serum  but  other  amboceptors  of  rabbit  serum  as 
well,  and  therefore  it  could  not  be  regarded  as  combining  with  such 
a  specific  receptor  as  the  cytophilic  group  of  the  amboceptor.  This 
work  was  confirmed  by  several  investigators,  including  Ehrlich  and 
Sachs,  who  agreed  with  Bordet  that  the  anti-amboceptor  does  not  com- 
bine with  the  cytophilic  group  but  offered  the  new  assumption  that  the 
combination  is  with  the  complementophilic  group.  Muir  and 
his  co-workers  have  studied  anti-amboceptors  extensively  and  find 
no  good  ground  for  accepting  the  later  interpretation  of  Ehrlich 
and  Morgenroth. 

Muir  offers  an  experiment  as  follows  to  illustrate  the  simple  action 
of  anti-amboceptor.  Two  tubes  are  marked  A  and  B.  Into  each  are 
placed  one  unit  of  cell  suspension  and  three  units  hemolytic  amboceptor 
(contained  in  rabbit  serum),  the  mixture  incubated  and  then  washed 
and  resuspended.  To  tube  A  is  added  0.3  c.c.  anti-amboceptor  (pre- 
pared by  injecting  rabbit  serum  into  guinea-pig),  and  to  tube  B  is 
added  0.3  c.c.  normal  inactivated  guinea-pig  serum.  The  mixtures  are 
again  incubated  and  washed;  to  each  tube  is  added  one  unit  comple- 
ment and  the  tubes  are  again  incubated.  Hemolysis  is  complete  in 
tube  B  but  is  absent  or  much  inhibited  in  tube  A,  thus  demonstrating 
the  inhibiting  effect  of  the  anti-amboceptor.  Such  an  anti-amboceptor 
as  is  here  illustrated  will  operate  only  against  amboceptors  contained 
in  rabbit  serum.  Similar  amboceptors  contained  in  goat  serum  would 
not  be  affected  by  the  anti-amboceptor  prepared  by  injecting  rabbit 
serum  into  guinea-pigs.  If  in  the  preceding  experiment  the  supernatant 
fluid  were  examined  after  the  first  incubation  it  would  be  found  that 
the  amboceptor  had  been  absorbed;  a  fact  also  illustrated  by  the 
hemolysis  in  tube  B.  Even  were  anti-amboceptor  and  amboceptor 
mixed  and  then  added  to  cells  the  amboceptor  would  not  be  prevented 
from  absorption.  If  the  supernatant  fluid  were  taken  after  the  last 
incubation  complement  would  be  found  free  in  tube  A  but  not  in  the 
full  original  amount,  as  can  be  shown  by  careful  titration.  The  anti- 
amboceptor  keeps  a  certain  amount  but  not  all  the  complement  from 
being  utilized.  The  converse,  however,  cannot  be  demonstrated,  that 
is  to  say,  complement  cannot  be  shown  to  inhibit  in  any  way  the  union 
of  amboceptor  and  anti-amboceptor.  Intricate  experiments  demon- 
strate, however,  that  the  union  of  amboceptor  and  anti-amboceptor  is 
loose  and  a  certain  amount  of  dissociation  may  occur  upon  the  addi- 
tion of  a  normal  serum  homologous  with  the  serum  which  contains 
the  amboceptor. 

Anti-complements. — As  has  been  indicated  above,  it  is  improbable 


138  THE  PRINCIPLES  OF  IMMUNOLOGY 

that  any  so-called  anti-complements  operate  differently  from  these  anti- 
amboceptors.  Numerous  substances  and  physical  conditions  are  anti- 
complementary  in  that  they  destroy  or  inhibit  the  action  of  complement. 
These  have  been  pointed  out  in  discussing  the  nature  of  complement 
and  must  be  considered  in  all  experiments  which  utilize  complement. 
It  has  been  suggested  that  in  the  interaction  between  amboceptor  and  anti- 
amboceptor  a  precipitate  is  formed  which  fixes  complement  and  that 
if  such  were  the  case  the  complement  should  not  be  recoverable.  Muir 
has  shown  that  it  is  possible  to  recover  complement,  as  we  have  pointed 
out  above.  Nevertheless,  even  such  a  form  of  fixation  may  permit  of 
dissociation,  and,  as  we  shall  show  in  discussing  complement  fixation, 
there  is  much  evidence  in  favor  of  the  view  that  the  action  of  these 
antilysins  is  dependent  upon  the  fixing  properties  of  precipitates. 

Physical  Hemolysis. — Hemolysis  is  produced  not  only  by  the 
serum  components  discussed  in  the  preceding  paragraphs  but  also  by 
chemical  and  physical  agents,  by  bacterial  products,  by  certain  vegetable 
poisons  and  by  venoms.  Studies  of  these  forms  of  hemolysis  are  of 
interest  not  only  because  of  their  intrinsic  value  but  also  because  they 
serve  to  throw  some  light  on  serum  hemolysis. 

The  necessity  for  using  an  isotonic  salt  solution  for  the  preservation 
of  erythrocytes  is  well  known  and  equally  well  known  is  the  fact  that 
reduction  of  salt  content  of  the  menstruum  beyond  a  certain  point  leads 
to  solution  of  hemoglobin,  which  in  distilled  water  is  seen  as  complete 
hemolysis.  This  is  not  merely  a  question  of  solubility  of  hemoglobin 
for  this  substance  is  soluble  in  physiological  salt  solution  to  the  same 
degree  as  in  distilled  water.  For  the  same  reason  it  is  not  a  matter 
of  simple  osmosis  of  the  hemoglobin.  Although  distilled  water  pro- 
duces swelling  of  the  cells  before  the  solution  of  hemoglobin  the 
rupture  of  the  cell  is  of  no  especial  importance  for  cells  may  be  cut 
into  pieces  in  physiological  salt  solution  without  hemolysis  appearing. 
From  experiments  of  Fischer  it  would  appear  that  the  hemoglobin  is 
held  in  combination  with  the  stroma  by  adsorption  and  that  the  action 
of  the  water  causes  a  physical  separation.  By  combining  fibrin,  a 
hydrophyllic  solid  colloid,  with  carmine,  a  hydrophobic  colloid  dye, 
Fischer  was  able  to  produce  phenomena  closely  resembling  hemolysis. 

Fragility  of  Erythrocytes. — The  corpuscles  of  different  animals 
differ  in  the  point  to  which  reduction  in  salt  concentration  of  the  sur- 
rounding menstruum  leads  to  hemolysis.  This  is  spoken  of  as  a  differ- 
ence in  resistance  to  hypotonic  salt  solution  or  a  difference  in  fragility. 
There  may  be  a  very  slight  difference  in  fragility  of  the  corpuscles  of 
different  individuals  of  the  same  species  and  diseased  conditions  may 
lead  to  well-marked  alterations.  In  man  a  simple  secondary  anemia  may 
lead  to  a  normal  or  reduced  fragility,  whereas  pernicious  anemia  leads  to 
increased  fragility.  Obstructive  jaundice  is  accompanied  by  decreased 
fragility,  whereas  familial  hemolytic  jaundice  shows  increased  fragility. 
In  the  anemia  of  animals  following  removal  of  the  spleen  there  is  a 
decrease  of  fragility  or,  in  other  words,  an  increase  of  resistance  to 
hypotonic  salt  solutions  and  also  to  other  hemolytic  agents. 


CYTOLYSINS  139 

Hemolysis  may  be  caused  by  other  physical  agents,  such  as 
freezing  (particularly  when  followed  by  thawing),  heat  of  62°  to  64°  C. 
in  the  case  of  mammalian  corpuscles  or  slightly  less  in  the  case  of 
cold-blooded  animals  and,  as  Rous  has  shown,  by  shaking. 

Chemical  Hemolysis. — The  influence  of  chemicals  on  hemolysis 
appears  to  be  a  factor  of  their  permeation  of  the  stroma.  Wells  states 
that  there  seem  to  be  two  types  of  permeating  substances,  one  such  as 
urea,  which  does  not  act  in  isotonic  solutions  of  sodium  chloride,  and 
the  other  such  as  ammonium  chloride,  which  acts  either  in  isotonic  or 
non-isotonic  solutions.  Hamburger,  as  quoted  by  Wells,  states  that 
erythrocytes  in  relation  to  organic  substances  are  (a)  impermeable 
for  sugars,  including  cane  sugar,  dextrose,  lactose,  arabinose  and  man- 
nose;  (b)  permeable  for  alcohols  in  inverse  proportion  to  the  number 
of  hydroxyl  groups  they  contain,  also  for  aldehydes  (except  paralde- 
hyde),  ketones,  ethers,  esters,  antipyrin,  amides,  urea,  urethan,  bile 
acids  and  their  salts;  (c)  slightly  permeable  for  neutral  amino-acids, 
such  as  glycocoll  and  asparagin.  In  relation  to  inorganic  substances,  not 
including  the  salts  of  fixed  alkalis,  the  erythrocytes  are  (a)  "  imperme- 
able for  the  cations  Ca,  Sr,  Ba,  Mg,  and  (b)  permeable  for  NH4  ions, 
for  free  acids  and  alkalis."  It  will  be  noted  that  certain  of  the  organic 
substances  for  which  the  cells  are  permeable  are  solvents  of  lipoids, 
particularly  those  lipoids  of  the  stroma,  cholesterol  and  lecithin,  a 
phenomenon  which  will  be  referred  to  again  in  discussing  venom 
hemolysis.  Other  chemical  hemolysins  include  veratrin,  digitalin, 
arseniuretted  hydrogen  (in  the  body  but  not  in  the  test  tube),  nitro- 
benzol  (important  in  denatured  alcohol  poisoning),  nitrites,  guaiacol, 
pyrogallol,  aniline  compounds,  alcoholic  extracts  of  tissues  and  products 
of  tissue  autolysis.  Salt  solution  extracts  of  various  organs  are 
hemolytic  and  have  been  called  organ  hemolysins.  These  bodies 
resist  boiling,  do  not  act  as  antigens,  hemolyze  at  37°  but  not  at  o°  C., 
are  not  increased  in  activity  by  complement  but  are  inhibited  by  the 
presence  of  serum.  Noguchi  has  studied  alcoholic  tissue  extracts  ex- 
tensively and  finds  them  also  hemolytic.  He  has  come  to  the  con- 
clusion that  the  active  elements  in  organ  hemolysis  are  soaps. 

Bacterial  Hemolysins. — Bacteria  may  by  their  growth  lead  to  suf- 
ficient acid  or  base  formation  in  the  media  as  to  make  the  latter 
hemolytic.  Of  equal  importance  is  the  fact  that  certain  bacteria  may 
produce  hemolytic  bodies  not  of  definitely  acid  or  alkaline  character, 
called  bacterial  hemotoxins.  These  substances  include  for  the  most 
part  the  products  of  pathogenic  organisms,  such  as  tetanolysin, 
staphylolysin,  streptolysin,  typholysin,  vibriolysin  (El  Tor  strain  of 
cholera) ,  anthrax-lysin  and  certain  other  less  important  forms.  Certain 
saprophytes  also  are  capable  of  producing  lysins,  as  for  example  mega- 
theriolysin,  proteus-lysin  and  the  lysin  of  bacillus  Welchii  and  others 
of  the  gas  gangrene  group.  An  important  bacterial  hemotoxin  is  that 
of  bacillus  pyocyaneus.  The  exact  nature  of  these  substances  is  not 
known,  but  Burckhardt  has  shown  that  staphylolysin  is  dialyzable, 
thermolabile,  ether  soluble  and  does  not  give  protein  or  biuret  reactions. 


I4o  THE  PRINCIPLES  OF  IMMUNOLOGY 

The  action  on  the  cells  is  independent  of  complement,  there  is  no  com- 
bination at  o°  C,  but  at  6°  C.  combination  occurs,  leading  to  hemolysis 
only  at  higher  temperatures.  It,  therefore,  seems  likely  that  these 
bodies  are  similar  to  toxins  with  a  special  affinity  for  erythrocytes. 
The  most  favorable  medium  for  developing  these  hemotoxins  is  broth, 
but  individual  organisms  require  special  conditions  in  the  broth  for 
maximal  production  of  hemotoxin.  The  development  of  the  toxin  fol- 
lows fairly  definite  curves  for  the  different  organisms.  For  example, 
staphylolysin  begins  to  appear  on  the  third  day,  reaches  a  peak  on  the 
fifth  day,  drops  on  the  sixth  day,  rises  again  on  the  eighth  day,  drops 
again  and  reaches  a  final  maximum  on  the  thirteenth  day.  Megatheri- 
olysin  reaches  a  maximum  on  the  seventh  day  and  almost  disappears 
by  the  fifteenth  day.  De  Kruif  has  recently  shown  that  streptolysin 
reaches  its  maximum  in  a  few  hours  and  has  almost  disappeared  by 
the  end  of  twenty-four  hours.  The  action  is  variable  for  different 
species  of  corpuscles;  staphylolysin  acts  powerfully  on  horse,  sheep 
and  other  bloods  but  only  slightly  on  human  and  goat  blood,  whereas 
megatheriolysin  acts  powerfully  on  human  blood  but  not  at  all  on  horse 
blood.  Nakayama  has  studied  the  streptolysin  and  finds  that  it  is 
filterable.  He  also  passed  the  organisms  through  two  species  of 
animals  and  finds  that  after  animal  passage  the  streptolysin  is  more 
actively  lytic  for  the  corpuscles  of  the  species  which  last  harbored  the 
organisms.  Streptolysin  unites  with  the  corpuscles  in  the  course  of 
hemolysis,  but  the  filtrate,  after  absorption  of  lysin,  remains  toxic  for 
mice.  Many  of  the  bacterial  hemotoxins  are  thermolabile,  being  de- 
stroyed at  60°  to  65°  C.,  but  others  are  resistant  to  temperatures  as 
high  as  100°  C.  The  bacterial  hemotoxins  are  active  in  vivo  as  well 
as  in  vitro  and  are  capable  of  producing  severe  anemias  and  even 
death.  An  intravenous  dose  of  2.0  c.c.  of  a  ten-day-old  filtered  broth 
culture  of  staphylococcus  produces  in  the  rabbit  marked  reduction  in 
hemoglobin  and  the  number  of  erythrocytes  and  may  cause  death  in 
six  or  seven  days.  It  is  probable  that  the  secondary  anemias  which 
often  follow  attacks  of  acute  infectious  disease  may  be  dependent  upon 
bacterial  hemotoxins.  Ford,  Lawrence  and  Williams  have  found  that 
cultivation  of  the  bacillus  Welchii  in  milk  leads  to  the  formation  of 
bacterial  hemolysins,  thus  disproving  the  opinion  previously  held 
that  the  hemolysis  in  gas  bacillus  infection  was  due  to  the  formation 
of  lactic  or  butyric  acid.  The  hemolysin  described  by  Ford  and  Law- 
rence is  relatively  stabile,  not  being  destroyed  until  a  temperature  of 
62°  or  63°  C.  has  been  reached.  It  has  other  characters  of  true  toxin 
in  that  it  is  digested  by  pepsin  and  hydrochloric  acid  as  well  as  by 
pancreatin.  It  is  precipitated  by  ethyl  alcohol.  It  is  antigenic,  and 
these  investigators  found  that  they  could,  by  immunization,  produce  an 
anti-hemolysin  or  anti-hemotoxin  in  titers  of  i-iooo,  1-1250. 

Vegetable  Hemolysins. — -Among  the  hemolytic  substances  of 
vegetable  origin  are  to  be  included  those  already  discussed  as  phyto- 
toxins,  namely  ricin,  abrin,  crotin,  robin,  phallin.  Crotin  and  phallin 
are  more  markedly  hemolytic  than  the  others,  which  are  rather  hemag- 


CYTOLYSINS  141 

glutinative  than  hemolytic.  The  phytotoxins  resemble  some  of  the 
bacterial  hemotoxins  in  that  they  may  serve  as  antigens  for  the  pro- 
duction of  antitoxins  but  differ  in  that,  as  a  rule,  they  are  thermostable. 
Both  groups  act  according  to  the  law  of  multiple  proportions.  Of 
considerable  importance  from  the  experimental  point  of  view  are  the 
saponins  "  a  closely  related  group  of  glucosides  found  in  at  least 
forty-six  different  families  of  plants"  (Wells).  They  are  thermo- 
stable, do  not  act  as  antigens,  have  a  fairly  definite  chemical  composi- 
tion and  are  in  these  particulars  to  be  separated  from  true  toxins.  They 
operate  injuriously  not  only  upon  the  erythrocytes  but  also  on  other 
body  cells,  especially  those  of  the  central  nervous  system.  Cholesterol 
and  lecithin  both  combine  with  saponin,  the  former  in  such  a  way  as 
to  prevent  hemolysis.  Therefore,  it  is  assumed  that  the  hemolytic 
action  of  saponin  is  dependent  upon  its  action  on  the  stroma  lipoids. 
Normal  serum  is  anti-hemolytic  for  some  of  the  saponins,  a  property 
which  may  be  slightly  increased  by  careful  immunization;  Robert  be- 
lieves this  increase  to  be  due  to  an  increase  of  blood  cholesterol. 

Venom  Hemolysins. — The  venom  hemolysins  or  hemotoxins  are 
found  in  different  amounts  in  all  venoms,  and  the  phenomenon  of  venom 
lysis  is  of  considerable  importance  not  only  because  of  its  scientific 
interest  but  also  because  of  its  employment  in  certain  clinical  tests. 
The  venoms  possess  not  only  lytic  but  also  hemagglutinative  properties, 
the  two  usually  being  present  in  inverse  ratio.  Flexner  and  Noguchi 
demonstrated  that  the  lysin  of  venoms  requires  activation  by  some 
substance  which  exercises  a  complementary  power.  They  found  that 
cobra  hemotoxin  dissolves  the  red  corpuscles  of  certain  animals  (ox, 
sheep  and  goat)  only  in  the  presence  of  serum,,  but  that  it  may  dis- 
solve other  erythrocytes  (dog,  guinea-pig,  man,  rabbit,  horse)  in  the 
absence  of  serum.  This  difference  is  probably  due  to  a  content  of 
activator  in  the  latter  cells,  which  activator  must  be  furnished  by  serum 
for  the  lysis  of  the  former  cells.  Kyes  found  that  he  could  extract 
an  activator  from  those  cells  which  do  not  require  serum  for  venom 
lysis  but  was  unable  to  do  so  in  the  case  of  those  cells  which  require 
serum  activation.  This  activating  substance  was  found  to  be  ether 
soluble.  Kyes  subsequently  found  that  lecithin  can  activate  venoms  and 
assumed  that  this  lipoid  constituted  the  bulk  of  the  activating  sub- 
stance. The  substance  in  serum  is  usually  active  only  after  the  serum 
has  been  heated,  but  with  some  sera  heating  is  not  necessary.  Kyes 
and  Sachs  believed  this  to  be  due  to  differences  in  the  nature  of  the 
lecithin  union  in  the  serum.  Kyes  mixed  cobra  poison  with  a  chloro- 
form solution  of  lecithin  and  obtained  a  substance  which  he  named 
cobra  lecithid  capable  of  activating  cobra  venom.  Von  Dungern  and 
Coca,  upon  investigating  the  subject,  came  to  the  conclusion  that  the 
cobra  venom  contains  a  ferment  capable  of  splitting  the  lecithin  so 
as  to  yield  certain  substances  such  as  oleic  acid  and  that  the  resulting 
hemolysis  is  due  to  the  activity  of  these  secondary  substances.  Noguchi 
holds  that  although  lecithin  exists  in  the  stroma  of  red  blood-cells  it 
is  not  present  in  a  form  available  for  venom  activation  and  that  the 


142  THE  PRINCIPLES  OF  IMMUNOLOGY 

degree  of  susceptibility  to  hemolysis  depends  upon  the  amount  of  such 
ether  soluble  activators  as  fatty  acids  (particularly  oleic  acid)  and 
their  soluble  soaps.  He  regards  fatty  acids,  neutral  fats  and  soluble 
soaps  as  endocellular  complement  and  assumes  certain  similarities  with 
serum  complement.  Certain  soap  serum  mixtures  were  found  to  be 
capable  of  completing  an  amboceptor  cell  mixture  but  numerous  objec- 
tions have  been  interposed  against  both  the  fact  and  the  interpretation 
so  that  at  the  present  time  there  is  no  good  ground  for  believing  that 
the  activator  of  cobra  lysin  is  a  true  complement  or  that  Noguchi's  soap 
mixtures  are  comparable  to  serum  complement.  If  the  activator  cannot 
be  regarded  as  complement,  the  venom  lysin  cannot  be  looked  upon 
as  an  amboceptor,  for  it  shows  no  specificity  and  does  not  require 
serum  complement  for  activation.  Kyes  in  a  recent  publication 
gives  what  may  be  regarded  as  the  modern  view  in  regard  to  venom 
lysis  as  follows : 

"  i.  There  is  present  in  all  venoms  a  hemolysin  existing  as  one  of 
a  number  of  distinct  toxins. 

"  2.  This  hemotoxin  effects  hemolysis  only  in  conjunction  with 
a  so-called  complementing  substance  which,  however,  may  be  found 
within  the  erythrocytes. 

"3.  The  reaction  between  the  hemotoxin  and  lecithin  is  essentially 
a  chemical  reaction  resulting  in  the  formation  of  a  complete  lysin. 

"  4.  This  complete  lysin  is  a  true  toxin  in  that  it  stimulates  the 
production  of  a  specific  antibody." 

Although  the  experiments  of  Zunz  and  Gyorgy  are  not  to  be  re- 
garded as  indicating  that  they  have  found  other  activating  substances 
for  cobra  venom  hemolysis,  nevertheless,  they  have  determined  that 
hemolytic  activity  of  cobra  venom  is  increased  by  certain  compounds  of 
protein  destruction,  including  certain  albumoses  and  amino-acids. 

The  hemolytic  property  of  cobra  venom  has  served  as  a  basis  for 
proposed  clinical  tests.  Calmette  noted  that  in  tuberculosis  the  blood 
contains  more  than  the  usual  amount  of  lecithin  and  that  small  amounts 
of  serum  of  such  patients  served  to  activate  cobra  venom  lysin.  The 
test  is  not  positive  in  more  than  78  per  cent,  of  tuberculous  patients  and, 
furthermore,  is  by  no  means  specific.  Similar  increases  of  lipoid  con- 
tent of  serum  have  been  found  in  certain  diseases  of  the  central  nervous 
system,  a  fact  leading  to  the  Much  and  Holzmann  psycho-reaction, 
which  also  is  not  specific.  Weil  has  maintained  that  in  syphilis  the 
corpuscles  are  more  resistant  to  venom  hemolysis  than  is  normal, 
except  in  the  earlier  stages  where  the  corpuscles  are  said  to  be  hyper- 
sensitive. As  time  has  passed  the  suggested  clinical  tests  have  not 
come  into  general  use  largely  because  of  lack  of  specificity.  Un- 
doubtedly the  blood  exhibits  alterations  in  lipoid  content  at  different 
times  in  various  diseases,  and  in  all  probability  there  is  a  parallel  altera- 
tion in  its  power  to  activate  venom  lysin,  but  no  one  disease  shows  this 
change  exclusively  or  even  constantly. 

Cytotoxins.  Specificity. — As  erythrocytes  may  act  as  antigenic 
substance,  so  may  other  body  cells.  The  antibodies  produced  by  injec- 


CYTOLYSINS  143 

tion  of  these  latter  cells  were  called  cytotoxins  by  Metchnikoff,  and 
the  name  has  been  retained  in  spite  of  the  fact  that  the  immune  bodies 
are  not  toxins  in  the  strict  sense  of  the  word  but  are  amboceptors 
similar  to  the  hemolytic  amboceptors.  It  was  thought  that  cytotoxins 
might  be  strictly  specific  for  the  antigenic  cells  of  different  organs 
within  the  same  species,  but  more  thorough  investigation  has  shown 
that  such  "  organ  specificity  "  is  not  demonstrable.  Hemolysins,  for 
example,  are  lytic  for  other  body  cells,  such  as  liver  and  kidney,  pro- 
vided these  cells  are  of  the  same  species.  Hepatolysins  and  nephrolysins 
are  also  active  as  hemolysins  within  the  species.  In  other  words, 
these  antibodies  are  species  specific  but  not  organ  specific.  It  may  be 
true  that  a  cytotoxin  acts-  more  especially  on  its  antigenic  organ  cells 
than  upon  other  cells,  as  is  maintained  by  Pearce  for  the  nephrolysins, 
but  the  action  is  not  exclusively  upon  the  antigenic  cells.  In  an  ex- 
tensive study  Pearce,  Karsner  and  Eisenbrey  were  unable  to  demon- 
strate any  strict  "  organ  specificity  "  by  means  of  cytolysis,  precipitation, 
agglutination  or  the  anaphylaxis  reaction.  In  this  work  the  organs  were 
washed  by  perfusion  with  large  amounts  of  salt  solution  and  thus  pre- 
pared for  injection.  Bell  has  pointed  out  that  even  the  most  careful 
perfusion  will  not  entirely  remove  the  blood  from  organs  and  therefore 
a  certain  amount  of  blood  must  be  injected  with  the  other  cellular 
antigen.  Nevertheless,  the  early  work  of  Landsteiner,  Metchnikoff  and 
of  Moxter  showed  that  spermatozoa  which  can  be  obtained  free  from 
blood  may  lead  to  a  spermatolysin  which  also  acts  as  a  hemolysin.  The 
amount  of  blood  injected  with  carefully- washed  organs  is  so  small 
that  it  can  have  but  little  antigenic  power,  too  little  to  be  consistent  with 
the  well-marked  hemolytic  power  of  the  cy  to  toxic  sera.  Recently, 
however,  Wilson  and  Oliver  have  absorbed  the  hemolysin  from  cyto- 
toxic  sera  by  means  of  erythrocytes  and  maintain  that  there  is  a  very 
definite  organ  specific  cytotoxin  contained  in  nephrolytic  serum  pre- 
pared by  immunizing  with  kidney  substance.  This  specific  nephrolysin 
can  be  removed  by  absorption  with  kidney  substance  but  not  with  other 
organs.  If  these  studies  are  extended  and  confirmed,  much  new  light 
may  be  thrown  on  the  subject  of  organ  specificity. 

In  spite  of  the  apparent  lack  of  strict  organ  specificity,  the  cytotoxins 
of  certain  types  of  cells  deserve  mention,  namely  those  resulting  from 
the  injection  of  leucocytes  and  of  crystalline  lens.  Following  a  brief 
communication  by  Delezenne  concerning  leucotoxins,  Metchnikoff 
studied  the  matter  by  injecting  guinea-pigs  with  material  from  the 
mesenteric  lymph-nodes  and  from  the  bone  marrow  of  rabbits.  The 
resulting  immune  serum  was  highly  toxic  for  guinea-pigs,  but  if  given 
in  sufficiently  small  doses  produced  first  a  marked  leucopenia,  fol- 
lowed in  several  days  by  a  leucocytosis.  This  was  confirmed  by  others 
who  used  for  injection  also  leucocyte  emulsion,  and  although  species 
specificity  was  strict,  the  cellular  specificity  was  not.  Lucatello  and 
Malon  were  able  to  obtain  a  serum  by  the  use  of  leucocytes  from  cases 
of  leucemia  and  treated  a  series  of  cases  with  this  serum.  The  leuco- 
cytes were  reduced  in  number  and  the  spleen  diminished  in  size,  but 


144  THE  PRINCIPLES  OF  IMMUNOLOGY 

there  was  no  permanent  improvement.  The  lack  of  cellular  specificity 
in  such  sera  is  an  a  priori  argument  against  their  use. 

Lens  Cyiotoxin. — The  injection  of  .crystalline  lens  leads  to  the 
formation  of  a  cytolysin  which  is  organ  specific  but  not  species  specific, 
similar  to  the  production  of  precipitins  by  lens  protein.  Such  a  cyto- 
toxin  prepared  by  the  use  of  the  crystalline  lens  of  the  dog  is  specific 
for  all  mammals,  birds  and  fish  and  will  not  act  upon  other  cells  from 
these  animals.  The  fact  that  the  injection  of  lens  into  animals  of  the 
same  species  or  even  into  the  same  individual  leads  to  the  production 
of  isocytotoxins  and  autocytotoxins  led  Romer  to  build  up  a  theory- 
concerning  the  origin  of  cataract.  He  suggested  that  the  constant 
absorption  of  lens  protein  from  the  normal  process  of  tissue  wear  and 
tear  leads  to  the  development  of  an  isocytotoxin  which  in  later  life 
produces  the  degeneration  of  the  Jens  seen  in  cataract.  If  such  were 
the  case  cataract  should  be  a  much  more  frequent  complication  of  age 
than  it  is  and  the  lens  should  be  a  soft  pulpy  organ  as  the  result  of  cyto- 
lysis.  Furthermore,  complement  is  not  available  in^the  fluids  of  the  eye 
and  the  cytolytic  amboceptor  is  not  to  be  completed  in  that  position. 
Other  theories  as  to  the  etiology  of  cataract  are  so  much  more  logical 
that  Romer 's  hypothesis  has  been  practically  abandoned. 

Aside  from  the  foregoing  example  of  isocytotoxin  and  autocytotoxin 
formation,  there  are  no  well-determined  illustrations  of  this  phenomenon 
except  for  the  demonstration  by  Metchnikoff  of  isospermatotoxins. 

Bacteriolysins. — The  death  and  solution  of  bacteria  in  the  processes 
of  resistance  to  disease  may  be  accomplished  by  the  activity  of  phago- 
cytic  body  cells  or  by  virtue  of  properties  of  the  blood  and  body  fluids 
similar  in  every  way  to  those  properties  which  lead  to  hemolysis.  In- 
deed, the  discovery  of  bacteriolysis  antedated  that  of  hemolysis  even  to 
the  point  of  understanding  the  essentials  of  its  mechanism.  Nuttall 
in  1888  demonstrated  that  fresh  normal  defibrinated  blood  has  the 
power  of  killing  bacteria.  He  set  up  a  series  of  tubes,  each  containing 
the  same  amount  (0.5  to  i.o  c.c.  defibrinated  blood)  and  added  to  each 
a  small  platinum  loopful  of  material  from  the  spleen  of  a  mouse  previ- 
ously inoculated  with  anthrax.  These  tubes  were  incubated  and  at  differ- 
ent time  intervals  gelatin  plates  were  made  from  the  tubes  and  a  control 
made  from  the  splenic  material.  This  showed  that  as  incubation  pro- 
ceeded the  bactericidal  activity  of  the  blood  became  apparent.  Buch- 
ner  confirmed  this  fact  in  1889  with  a  slightly  different  method, 
whereby  a  larger  amount  of  blood  and  bacteria  were  mixed  in  one 
container  and  incubated  and  small  standard  amounts  withdrawn  by 
pipette  and  plated.  It  was  found  that  if  the  blood  were  heated  or 
allowed  to  stand,  its  bactericidal  power  was  lost  and  Buchner  named 
the  thermolabile  element  alexin.  He  believed  it  to  be  of  the  nature 
of  a  ferment,  suggested  that  it  might  originate  in  body  cells,  possibly 
leucocytes,  and  recognized  the  fact  that  it  is  not  specific. 

The  Pfeiffer  Phenomenon. — The  next  important  advance  appeared 
in  the  studies  of  Pfeiffer  and  his  co-workers,  who,  in  1893,  1894  and 
subsequently,  published  the  details  of  what  we  now  speak  of  as  the 


FlG.  15. — Stages  in  lysis  of  cholera  vibrios, 
showing  the  reduction  to  large  coccoid 
forms  before  final  solution.  (Modified 
from  Pfeiffer  and  Friedberger,  Lehrbuch 
der  Mikrobiologie.  Jena,  1919). 


• , 


'V7 


CYTOLYSINS  145 

Pfeiffer  phenomenon.  These  discoveries  were  incident  to  the  investi- 
gation of  immunity  to  cholera  spirilla.  The  method  is  essentially  that 
of  studying  the  changes  taking  place  in  the  spirilla  following  intraperi- 
toneal  injection  in  guinea-pigs.  If  the  guinea-pig  had  survived  preceding 
inoculations  and  had  thereby  developed  immunity  the  injection  of  or- 
ganisms was  followed  by  loss  of  their  motility,  transformation  into 
oval  translucent  granules  and  finally  disappearance  of  the  bacteria  with 
complete  recovery  of  the  animal.  If  the  spirilla  were  of  only  low 
degree  of  virulence  the  same  phenomenon  could  be  observed  in  a 
normal  animal,  but  if  the  animal  were  highly  immune  it  could  survive 
doses  of  virulent  organisms  much  greater  than  those  fatal  for  normal 
guinea-pigs.  It  was  found  that  the  simultaneous  intraperitoneal  injec- 
tion of  serum  from  an  immune  pig  and  of  spirilla  into  a  normal  pig 
served  to  protect  the  animal  and  that  this  protection  could  be  conferred 
as  well  by  heated  as  by  non-heated  immune  serum.  The  mechanism  in 
all  cases  was  the  same  and  not  dependent  upon  phagocytic  activity. 
Furthermore,  the  protection  was  found  to  be  specific.  Pfeiffer  was 
unable  to  demonstrate  the  phenomenon  in  vitro  (hanging  drop  prepara- 
tion) and  therefore  assumed  that  some  substance  provided  by  the  peri- 
toneal endothelium  served  to  activate  the  bacteriolytic  process. 

In  the  demonstration  of  the  Pfeiffer  phenomenon  it  is  necessary  to  have 
a  series  of  fairly  young  guinea-pigs  of  about  200  grams  in  weight  and  a-  culture 
of  cholera  spirilla  whose  virulence  is  well  established,  because  the  virulence  of 
the  organisms  plays  quite  as  important  a  part  as  their  number.  The  immune 
serum  may  be  produced  in  the  rabbit,  goat  or  other  animal  by  repeated  inocula- 
tion with  the  organisms.  The  organisms  may  be  injected  in  measured  volumes 
of  broth  cultures  or  of  saline  suspensions  of  agar  cultures ;  they  may  also  be 
measured  by  weight  by  the  use  of  a  standard  platinum  loop  which  takes  up 
approximately  0.002  gm.  organisms.  The  immune  serum  is  diluted  as  indicated 
in  the  following  protocol  and  the  bacteria  and  serum  are  injected  simultaneously. 
Peritoneal  fluid  is  withdrawn  at  intervals  of  10,  20,  30,  45,  60  minutes,  the 
intervals  being  altered  as  circumstances  indicate.  The  withdrawal  is  by  means 
of  drawn  out  capillary  pipettes  introduced  into  the  belly  cavity  through  a 
small  incision  in  the  skin.  The  material  may  be  examined  in  a  hanging  drop  or 
may  be  spread  and  stained  by  the  ordinary  bacterial  dyes.  A  protocol  from 
Pf eiffer's  own  work  follows  : 

PFEIFFER  PHENOMENON 

Weight  of  0986  of  Dose  of 

guinea-pig  spirilla  immune  Examination  of 

in  grams  in  grams  serum  in  c.c.  Result  peritoneal  fluid 

320  o  002  0.05  Lives  After  15  minutes,  only  gran- 

ules present. 

240  0.002  0.02  Lives  After  20  minutes,  only  gran- 

ules present. 

200  0.002  0.006  Lives  Sterile  after  35  minutes. 

220  0.002  0.003  Lives  After  25  minutes,  numerous 

granules,  isolated,  non- 
motile  spirilla.  After  I 
hour  practically  sterile. 

220  0.002  o.ooi  Died  during  After  25  and  50  minutes, 

night  numerous  granules  but 

also  numerous  active  spir- 
illa. After  loo  minutes, 
only  active  spirilla. 

10 


146  THE  PRINCIPLES  OF  IMMUNOLOGY 

PFEIFFER  PHENOMENON—  (Continued) 

Weight  of          Dose  of  Dose  of 

guinea-pig  spirilla  immune  Examination  of 

in  grams  in  grams  serum  in  c.c.  Result  peritoneal  fluid 

230  0.002  0.0005  Died  during  After   25    minutes,    a    few 

night  granules,  numerous  active 

spirilla.  Progressive  in- 
crease of  spirilla. 

200  0.002  0.2  c.c.  Died  during  After  25  minutes,  few  gran- 

normal  night  ules,      numerous      active 

guinea-pig  spirilla.      Autopsy     after 

serum  as  several  hours  snowed  pus 

control  on    the    liver,    numerous 

spirilla  mostly  free  in  exu- 
date,  with  granules  both 
free  and  within  cells. 

In  the  foregoing  experiment  it  is  seen  that  0.003  c.c.  immune  serum 
serves  to  protect  a  guinea-pig  of  about  200  grams  from  an  otherwise 
fatal  dose  of  cholera  spirilla.  Pfeiffer  used  this  method  to  titrate  bac- 
teriolytic  sera  and  in  this  case  would  have  indicated  the  serum  as 
containing  in  each  cubic  centimeter  333  immune  units. 

Bacteriolysis  in  Vitro. — Further  study  of  the  phenomenon  more 
particularly  by  Metchnikoff  and  by  Bordet  led  to  the  discovery  that  the 
process  may  be  demonstrated  in  vitro,  in  spite  of  Pf eiffer's  failure  to  do 
so.  Metchnikoff  was  able  to  produce  lysis  of  spirilla  in  hanging  drop 
preparations  by  adding  to  the  mixture  of  spirilla  and  immune  serum  an 
extract  of  leucocytes,  thus  offering  evidence  in  favor  of  the  influence 
of  leucocytes  in  destruction  of  bacteria.  Bordet  demonstrated  that 
although  the  activity  of  the  immune  serum  is  destroyed  by  heat  of 
50°  C.  to  60°  C.,  the  serum  may  be  rendered  active  again  by  the  addi- 
tion of  a  small  amount  of  fresh  serum,  an  amount  of  fresh  serum  in 
itself  incapable  of  producing  bacteriolysis.  He  found  that  the  speci- 
ficity of  the  immune  serum  resides  in  a  substance  which  he  later  named 
the  sensitizer  (the  Ehrlich  amboceptor).  The  alexin  of  Buchner 
(complement)  was  found  to  exhibit  no  specificity  and  was  not  increased 
by  immunization.  In  the  course  of  these  studies  Bordet  found  that 
the  corpuscles  in  the  fresh  normal  guinea-pig  serum  were  agglutinated 
by  the  immune  goat  serum  and  that  the  spirilla  were  often  likewise 
agglutinated.  Suspecting  that  if  both  blood-cells  and  bacteria  are 
agglutinable,  the  blood-cells  might  be  the  subjects  of  lysis  as  well  as 
are  bacteria,  Bordet  was  led  to  the  discovery  of  the-  phenomenon  of 
hemolysis.  The  studies  of  Toitsu,  Matsunami  and  Kolmer  would  indi- 
cate that  all  bacteriolysis  is  not  necessarily  dependent  upon  the  activity 
of  complement,  for  they  found  that  anti-meningitis  sera  which  were 
freed  from  complement  possessed  bactericidal  properties.  Ecker  has 
made  similar  observations  in  regard  to  a  serum  specifically  bacteriolytic 
for  the  diphtheria  bacillus.  Nevertheless,  Ecker  found  that  the  addi- 
tion of  complement  increases  the  bacteriolytic  action  of  this  serum. 

The  Pfeiffer  phenomenon  was  found  applicable  to  bacteria  other 
than  the  cholera  spirilla,  including  particularly  the  typhoid  bacillus, 
paratyphoid,  dysentery  and  colon  bacillus.  With  these  organisms  the 
phenomenon  proceeds  more  slowly  than  with  cholera  spirilla.  Were 


CYTOLYSINS  147 

no  simpler  means  available,  the  Pfeiffer  phenomenon  might  well  serve 
as  a  laboratory  method  of  identifying  cultures  of  the  bacteria. 

Wright's  Method  for  Bacteriolysis. — In  the  course  of  subsequent 
studies,  other  methods  of  investigation  of  bacteriolysis  have  been  de- 
vised, those  of  Wright,  of  Neisser  and  Wechsberg  and  of  Buxton  de- 
serving especial  mention.  Wright  exercised  his  usual  ingenuity  in 
attacking  this  problem  and  devised  two  methods,  one  by  dilution  of 
serum  and  the  other  by  dilution  of  the  culture  of  organisms.  For  the 
collection  of  serum  he  used  the  Wright  pipette  such  as  is  employed 
for  determining  opsonic  content  of  serum.  The  serum  was  diluted 
with  different  amounts  of  bouillon.  The  culture  was  mixed  with 
melted  gelatine  and  to  measured  amounts  of  this  mixture  was  added 
the  proper  amount  O'f  serum  dilution.  The  final  mixtures  were  incu- 
bated in  capillary  pipettes  for  two  to  three  days  at  22°  C,  then  placed 
under  low  magnification  of  the  microscope  and  the  number  of  colonies 
in  the  pipettes  determined.  In  the  second  method  the  culture  was 
diluted  in  varying  amounts  of  broth  by  means  of  a  specially  con- 
structed capillary  pipette  and  the  suspension  blown  into  a  watch  glass. 
The  culture  dilutions  were  mixed  with  a  standard  amount  of  serum 
and  incubated  in  special  pipettes.  If  the  serum  was  insufficient  to  kill 
all  the  organisms,  there  was  bacterial  growth,  and  the  medium  became 
cloudy.  Having,  by  previous  plating,  determined  the  number  of  organ- 
isms in  a  given  bulk  of  broth  culture,  it  was  possible  to  determine  how 
many  organisms  could  be  killed  by  the  standard  amount  of  serum. 
The  outlines  of  these  methods  are  given  because  of  the  ingenuity  dis- 
played and  the  exact  information  gained,  although  at  the  present  time 
they  are  not  extensively  employed. 

The  Neisser- Wechsberg  Phenomenon. — The  Neisser  and  Wechs- 
berg method  was  described  almost  contemporaneously  with  that  of 
Wright.  They  mixed  inactivated  serum  dilutions  in  test  tubes  with 
either  broth  cultures  or  salt  solution  suspensions  of  organisms,  added 
complement  and  incubated.  Definite  amounts  of  these  mixtures  were 
added  to  melted  solid  culture  media,  such  as  agar,  and  plates  poured. 
After  incubation  of  the  plates,  the  colonies  were  counted  and  the  bacte- 
riolytic  activity  of  the  serum  thus  determined.  A  protocol  taken  from 
the  studies  of  Neisser  and  Wechsberg  will  serve  to  illustrate  the  method. 


Tubes 

I 

NEISSER-  WECHSBERG  PHENOMENON 

Amount                         Inactivated             Fresh  guinea- 
of  culture                     immune  serum            pig  serum 

1/5000  c.c.   of   a   24- 

Number  of  colonies 
on  plates 

hour     broth     culture 

of  vibrio  Metchnikovi       i.o  c.c. 

0.3  c.c. 

Many  thousands  ; 

2 

0.5  c.c. 

0.3  c.c. 

Many  thousands 

3 

0.25  c.c. 

0.3  c.c. 

Many  thousands 

4 

O.I    C.C. 

0.3  c.c. 

Few  hundred 

0.05  c.c. 

0.3  c.c. 

About  100 

6 

0.025  c.c. 

0.3  c.c. 

About  50 

7 

O.OI    C.C. 

0.3  c.c. 

None 

8 

0.005  c.c. 

0.3  c.c. 

None 

9 

0.0025  c.c. 

0.3  c.c. 

About  100 

10 

O.OOI    C.C. 

0.3  c.c. 

Many  thousands 

ii 

"                        0.0005  c.c. 

0.3  c.c. 

Many  thousands 

148  THE  PRINCIPLES  OF  IMMUNOLOGY 

NEISSER-WECHSBERG   PHENOMENON  (Continued) 

Amount  Inactivated  Fresh  guinea-  Number  of  colonies 

Controls  of  culture  immune  serum  pig  serum  on  plates 

1  1/5000  c.c.  ...  ...  Many  thousands 

2  1/5000  c.c.  o.oi  c.c.  . . .  Many  thousands 

3  o.oi  c.c.  . . .  None 

4  1/5000  c.c.  ...  0.3  c.c.  Many  thousands 

5  ...  1.0  c.c.  None 

The  broth  culture  is  so  diluted  that  0.5  c.c.  are  added  to  each  tube.  All  tubes 
are  made  up  to  constant  volume  with  0.85  per  cent,  salt  solution.  Incubation  is 
for  three  hours  at  37°  C,  after  which  five  drops  from  each  tube  are  added  to 
a  tube  of  melted  agar  for  plating. 

The  Neisser-Wechsberg  method  not  only  presents  a  means  of  work- 
ing with  bactericidal  sera  but  also  demonstrates  both  the  necessity  for 
the  presence  of  complement  to  complete  the  bactericidal  amboceptor 
and  the  appearance  of  inhibition  zones  in  the  stronger  concentra- 
tions of  the  immune  serum.  Neisser  and  Wechsberg  interpreted  the 
inhibition  zone  as  illustrating  what  they  called  "  complement  devia- 
tion," a  term  frequently  used  incorrectly  as  synonymous  with  com- 
plement fixation.  They  believed  that  if  an  excess  of  amboceptor  units 
is  present,  a  certain  number  of  these  units  will  combine  with  the  avail- 
able complement  units,  thus  leaving  a  number  of  amboceptor  units 
unsaturated  with  complement.  The  amboceptor  is  present  in  amounts 
too  large  to  be  entirely  absorbed  by  the  antigenic  bacteria  and  therefore 
it  is  assumed  that  a  certain  number  of  the  free  amboceptor  units 
combine  with  a  number  of  complement  units,  thus  preventing  a  suf- 
ficient amount  of  complement  to  combine  with  the  amboceptor  units 
already  absorbed  by  the  bacteria  for  the  process  of  bacteriolysis.  In 
tubes  four  to  nine  of  the  preceding  protocol  the  amboceptor  units  and 
bacteria  are  closely  enough  balanced  to  ensure  complete  absorption 
of  amboceptor  and  thus  permit  of  full  action  of  complement;  there 
being  no  free  amboceptor,  there  is  no  "  deviation  "  of  complement. 
Except  for  the  possible  evidence  afforded  by  the  Ehrlich  and  Sachs 
experiment  (see  page  125)  there  is  no  other  experimental  evidence  sup- 
porting the  view  that  free  amboceptor  may  enter  into  combination 
with  complement.  Gay  has  suggested  that  the  inhibition  may  be  due  to 
precipitation  by  the  interaction  of  the  immune  serum  and  bacterial 
protein  which  may  have  gone  into  solution,  the  precipitate  operating 
to  fix  complement  and  prevent  its  combination  with  bacteriolytic  am- 
boceptor. Whilst  precipitation  may  undoubtedly  be  of  significance  in 
this  connection,  we  are  of  the  opinion  that  the  resemblance  to  col- 
loidal reactions  as  described  in  connection  with  precipitation  and  agglu- 
tination, wherein  excess  of  one  colloid  may  prevent  the  occurrence  of 
precipitation  or  flocculation,  offers  an  equally  satisfactory  explanation 
ifor  the  Neisser-Wechsberg  phenomenon  and  that  we  are  therefore 
justified  in  regarding  the  reaction  as  illustrating  "  inhibition  zones  " 
where  the  concentration  of  amboceptor  is  great.  The  failure  of  bac- 
teriolysis in  tubes  eleven  and  twelve  is  due,  of  course,  to  insufficient 
amount  of  amboceptor.  The  control  tubes  show  that  neither  ambo- 
ceptor nor  complement  alone  is  capable  of  producing  bacteriolysis. 
Buxton's  Method  for  Bacteriolysis. — Buxton  determined  that 


CYTOLYSINS  149 

active  immune  serum  shows  the  same  inhibition  zones  and  also  simplified 
the  method.  By  allowing  the  original  tubes  to  incubate  twenty-four 
hours  at  37°  C,  the  degree  of  clouding  of  the  medium  by  bacterial 
growth  gives  an  excellent  indication  of  the  degree  of  bacteriolysis.  He 
found  that  normal  rabbit  serum  shows  bacteriolytic  powers  in  strong 
concentration,  gradually  diminishing  as  dilution  proceeds.  Thus  the 
low  titer  normal  amboceptor  fails  to  show  inhibition  zones,  as  is  true 
of  low  titer  agglutinins  and  precipitins.  A  protocol  from  Buxton's 
work  shows  the  difference  in  activity  of  normal  serum  and  immune 
serum  as  well  as  the  correspondence  between  the  results  of  plating  and 
observation  of  original  tubes. 

BUXTON  EXPERIMENT 

Dilution  Count  of  colonies  on  plates  Observation  of  original  tubes 

of  sera  Normal  serum  Immune  serum         Normal  serum  Immune  serum 

i  o  Many  thousand  Clear  Cloudy 

-2  o  Many  thousand  Clear  Cloudy 

-5  2  Many  thousand  Clear  Clear 

-20  2500  4-5000  Cloudy  Clear 

-40  Many  thousand  4-5000  Cloudy  Clear 

-80  Many  thousand  Many  thousand  Cloudy  Cloudy 

-100  Many  thousand  Many  thousand  Cloudy  Cloudy 

Teague  and  McWilliams  have  confirmed  the  work  of  Buxton  and 
others  showing  that  normal  rabbit  serum  is  capable  of  killing  large 
numbers  of  typhoid  and  paratyphoid  bacilli,  but  that  the  sera  of  rabbits 
highly  immunized  against  these  organisms  do  not  kill  these  bacilli. 
These  investigators  have  emphasized  further  that  the  normal  bacteri- 
olytic activity  of  rabbit  serum  for  typhoid  and  paratyphoid  bacilli  is 
not  materially  altered  by  immunization.  In  human  typhoid  fever  the 
blood  serum  normally  shows  bacteriolytic  activity,  but  in  spite  of  this  bac- 
teria multiply  in  the  tissues  apparently  because  the  lymph  does  not  pos- 
sess bacteriolytic  powers.  Stone  more  recently  made  similar  observations 
but  found  further  that  fresh  immune  typhoid  serum  in  vivo  has  appar- 
ently a  high  bactericidal  power,  while  fresh  normal  serum  in  vivo 
has  no  protective  power.  Typhoid  bacilli  disappear  more  quickly  from 
the  organs  of  immune  animals  than  from  normal  animals,  but  macerated 
organs  from  immune  animals,  cut  sections,  or  their  extracts  are  not 
bactericidal  even  on  the  addition  of  fresh  immune  serum.  This  work 
indicates  that  the  destruction  of  typhoid  bacilli  in  the  immune  animal 
is  due  to  some  interaction  between  the  tissue  cells  and  plasma  in  vivo 
or  other  unknown  factor. 

The  Bioscopic  Method  for  Bacteriolysis. — Neisser  and  Wechsberg 
also  devised  the  so-called  bioscopic  method  of  studying  bacteriolysis. 
They  took  advantage  of  the  fact  that  living  cells  possess  the  power  of 
converting  methylene  blue  into  its  colorless  leucobase.  By  careful 
adjustment  of  the  number  of  bacteria  it  was  possible  to  mix  the  various 
agents  together,  add  a  very  dilute  alcoholic  solution  of  methylene  blue, 
cover  with  paraffin  and  incubate.  The  degree  of  decolorization  indi- 
cates the  relative  amount  of  bacterial  growth. 

Summary  of  Cytolysis. — In  summary  it  may  be  said  that  the  phe- 


150  THE  PRINCIPLES  O*F  IMMUNOLOGY 

nomenon  of  cytolysis  represents  a  general  biological  phenomenon  ap- 
plicable to  vegetable  cells,  exemplified  by  bacteria,  and  also  to  a  wide 
variety  of  animal  cells.  In  both  kingdoms  there  is  a  marked  species 
specificity  exhibiting,  as  do  other  immune  processes,  the  phenomenon 
of  group  reactions.  In  so  far  as  bacteriolysis  is  concerned,  inhibition 
zones  appear,  apparently  similar  to  the  inhibition  zones  of  precipitation 
and  agglutination.  Two  bodies  interact  to  produce  cytolysis,  a  ther- 
mostable body,  the  amboceptor  or  sensitizer,  which  may  be  increased 
by  immunization,  and  a  thermolabile  body,  the  complement  or  alexin, 
which  does  not  react  to  immunization.  The  amboceptor  appears  to 
act  by  preparing  the  antigenic  cells  for  the  lytic  action  of  the  com- 
plement rather  than  by  furnishing  a  two-armed  link  between  cell  and 
complement.  The  reaction  takes  place  more  nearly  according  to  the 
physical  chemical  laws  of  colloidal  reactions  than  the  simpler  laws  of 
reactions  between  inorganic  chemicals.  The  protection  afforded  an 
animal  which  possesses  bacteriolytic  immune  bodies  is  obvious,  and 
the  role  these  bodies  play  in  natural  and  acquired  immunity  to  disease 
must  be  of  great  importance. 


CHAPTER  VII 
CELLULAR  RESISTANCE 

PHAGOCYTOSIS. 

INTRODUCTION. 

THE  PROCESS  OF  PHAGOCYTOSIS. 

MUTUAL  APPROACH    (CHEMOTAXIs). 
INGESTION. 
DIGESTION. 

CELLS  WHICH  PARTICIPATE. 
FUNCTIONS   OF  THE  PROCESS. 
EXPERIMENTAL   DEMONSTRATION. 
MECHANISM  OF  PHAGOCYTOSIS. 

PHYSICAL  BASIS. 
OPSONINS. 

INTRODUCTION  AND  DEFINITION. 
EXPERIMENTAL   DEMONSTRATION. 
NORMAL  OPSONINS. 
IMMUNE  OPSONINS. 

B  ACTERIOTROPI N  S . 

OPSONINS  FOR  CELLS  OTHER  THAN  BACTERIA. 
SPECIFICITY  AND  OTHER  CHARACTERS. 
INFLUENCE  OF  PHAGOCYTE  AND  INGESTED  ELEMENTS. 
RELATION  OF  BACTERIAL  VIRULENCE. 
INFLUENCES  OPERATING  UPON  PHAGOCYTES. 
ANALYSIS  OF  MECHANISM   OF  PHAGOCYTOSIS. 
OTHER   MANIFESTATIONS   OF  CELLULAR  RESISTANCE. 

INTRODUCTION. 
THE  LEUCOCYTES. 

BACTERICIDAL  EXTRACTS. 

LEUCOCYTE  ANTIBODY. 
LEUCOCYTE  ENZYMES. 

LEUCOCYTE  EXTRACTS  FOR  THERAPEUSIS. 
SPECIFIC    HYPERLEUCOCYTOSIS. 
THE  LYMPHOCYTES. 
THE   PLATELETS. 
THE  INFLUENCE  OF  INFLAMMATION. 

PHAGOCYTOSIS 

Introduction. — MetchnikofT  has  defined  the  phagocyte  as  a  cell 
capable  of  ingesting  foreign  bodies.  Similarly  the  process  of  phago- 
cytosis can  be  referred  to  as  the  process  of  ingestion  of  foreign  bodies 
by  a  cell.  In  the  study  of  unicellular  organisms  and  of  certain  lower 
forms  of  multicellular  organisms  it  has  been  found  that  the  process 
of  phagocytosis  is  an  important  means  of  obtaining  nutrition.  That 
such  a  simple  process  could  have  any  bearing  on  the  resistance  of 
vertebrates  to  disease  was  not  pointed  out  for  many  years.  The  very 
earliest  study  of  bacteriology  and  immunity  led  to  the  knowledge  of  the 
fact  that  the  injection  of  bacteria  into  animals  led,  under  favorable 
conditions,  to  the  disappearance  of  these  bacteria.  The  investigation 
of  the  cause  of  this  disappearance  resulted  first  in  the  belief  that  it  was 
due  to  solution  of  the  bacteria  by  body  fluids,  more  especially  the  blood. 
Certain  early  investigators  had  noticed  that  following  such  injections 
of  bacteria  the  bacteria  might  appear  within  tissue  cells,  but  Panum 

151 


152  THE  PRINCIPLES  OF  IMMUNOLOGY 

was  the  first  to  interpret  the  phenomenon  in  an  immunological  sense. 
He  pointed  out  that  the  penetration  of  bacteria  into  living  cells,  as 
previously  maintained  by  Birch-Hirschfeld,  probably  had  much  to  do 
with  the  disappearance  of  bacteria  following  injection.  Subsequently, 
it  was  shown  that  bacteria  do  not  penetrate  into  cells  but  rather  are 
taken  up  by  the  cells.  This  work  of  Panum  did  not  lead  to  any  direct 
result  in  the  study  of  immunology,  for  Metchnikoff  in  his  early  work 
on  the  subject  was  ignorant  of  it.  Roser  had  also  stated  that  according 
•to  his  opinion  the  immunity  of  healthy  animals  and  plants  against  in- 
fectious organisms  rests  upon  (a)  the  relative  salt  content  of  their 
fluids  and  (b)  the  capacity  of  their  contractile  cells  to  take  up  the 
invading  organisms.  Roser,  however,  did  not  support  this  statement  in 
later  studies,  and  again  Metchnikoff  was  ignorant  of  this  work  when 
he  took  up  his  great  work  on  phagocytosis.  Metchnikoff  had  studied 
extensively  the  nutrition  of  certain  of  the  lower  forms  of  animal  and 
vegetable  life  and  also  their  defenses  against  the  invasion  of  harmful 
parasites.  From  this  work  he  was  led  to  the  conclusion  that  the  defense 
of  higher  animals  depends  in  great  part  upon  the  phenomenon  of 
phagocytosis.  This  statement  was  magnified  into  a  conflict  between 
the  so-called  cellular  theories  of  immunity  and  the  humoral  theories 
of  immunity.  At  the  present  time,  however,  such  a  conflict  does  not 
exist  because  the  two  theories  of  immunity  are  in  perfect  harmony  with 
one  another,  and  it  is  known  that  they  are  dependently  interrelated. 

The  process  of  phagocytosis  involves  three  steps;  first,  the  ap- 
proach of  the  cell  and  the  material  to  be  taken  up ;  second,  the  ingestion 
of  the  material,  and,  third,  the  destruction  of  such  material  as  may  be 
dissolved  by  the  digestive  fluids  of  the  cell.  The  problem  of  the 
approach  of  the  cell  and  material  to  be  ingested  is  one  fundamentally 
of  irritability.  The  irritability  of  living  tissue  is  in  response  to  certain 
stimuli  and  such  stimuli  include  chemical,  thermal,  osmotic,  photic, 
mechanical  and  other  physical  agents.  In  the  early  studies  of  the 
physiology  of  stimulation  the  response  of  a  cell  to  a  stimulus  was 
believed  to  be  governed  by  the  Weber-Fechner  law,  which  states  that 
the  intensity  of  sensation  varies  with  the  logarithm  of  the  intensity 
of  the  stimulus  or,  in  other  words,  as  the  stimulus  increases  by  geometric 
progression  the  response  increases  by  arithmetical  progression.  This 
law  has  been  found  by  further  study  to  be  untenable,  for  it  has  been 
shown  that  logarithmic  functions  are  not  applicable  to  very  strong 
stimuli.  In  phagocytosis  chemical  stimuli  are  the  most  important,  and 
the  response  to  such  stimuli  is  referred  to  as  chemotaxis. 

Mutual  Approach  (Chemotaxis). — Chemotaxis  may  be  positive  or 
negative,  according  to  whether  it  attracts  the  two  bodies  or  repels  them. 
Such  attraction  or  repulsion  does  not  depend  essentially  on  the  acidity 
or  alkalinity  of  the  medium  but  does  depend  in  certain  measure  upon 
its  concentration.  Not  only  does  variability  of  concentration  play  a 
part,  but  the  adaptability  of  the  cell  itself  is  of  importance ;  for  example, 
myxomycetes  plasmodia  exhibit  negative  chemotaxis  in  the  presence 
of  sugar  in  certain  concentrations,  but  after  the  organism  becomes 


CELLULAR  RESISTANCE  153 

accustomed  to  the  presence  of  the  sugar  a  positive  chemotaxis  appears. 
The  lower  animal  and  vegetable  cells  exhibit  a  certain  amount  of  selec- 
tion in  the  material  which  they  take  up,  and  the  leucocytes  of  higher 
organisms  may  in  a  similar  manner  exhibit  selectiveness.  According 
to  our  present-day  physical  conception  of  the  activity  of  living  proto- 
plasm, this  selectiveness  would  depend  in  all  probability  upon  varying 
sensibility  to  chemotactic  influences  or  variation  in  the  intensity  of  the 
chemotactic  stimuli. 

Ingestion  of  Foreign  Body. — The  actual  ingestion  of  the  foreign 
body  depends  upon  the  motility  of  the  cell  protoplasm,  and  this  motility, 
of  course,  is  a  function  of  the  irritability  of  the  protoplasm.  Such 
motility  determines  the  ameboid  movement  of  the  cell  to  the  material 
to  be  ingested.  Having  approximated  itself  to  the  foreign  material, 
the  cell  throws  out  pseudopodia  in  such  a  fashion  as  to  encircle  the 
foreign  body ;  as  opposite  pseudopodia  meet  the  cell  resumes  in  so  far 
as  possible  its  normal  form  and  the  material  is  enclosed  in  the  cell 
protoplasm.  These  two  stages  in  the  process  of  phagocytosis  have 
been  reduplicated  in  experiments  with  non-living  material. 

Digestion. — The  third  stage  of  phagocytosis  is  the  digestion  of  the 
foreign  material.  Such  digestion  is  accomplished  by  secretions  which 
are  poured  out  by  the  cell  protoplasm  so  as  to  constitute  a  small  area 
of  fluid  about  the  ingested  particle.  By  staining  with  dyes  which  show 
the  acid  reaction  it  has  been  found  that  although  the  cell  protoplasm 
does  not  show  acidity  the  fluid  within  the  digestive  vacuole  is  definitely 
acid  in  character.  Attempts  to  extract  this  digestive  fluid  from  protozoa 
have  not  been  highly  successful,  but  Mouton  was  able  to  extract  from 
a  symbiotic  culture  of  amebse  and  colon  bacilli  an  enzyme  which  is 
feebly  proteolytic.  This  enzyme  is  capable  of  digesting  colon  bacilli 
which  have  been  killed  but  does  not  act  upon  living  colon  bacilli.  The 
intracellular  digestion  of  these  particles  depends  upon  their  solubility 
by  the  digestive  fluids.  The  cell  may  take  up  insoluble  particles,  in 
which  case  the  particles  remain  within  the  cells  or  are  extruded  with 
the  excreta  of  the  cells. 

Types  of  Phagocytic  Cells. — It  is  incorrect  to  think  of  leucocytes 
as  identical  with  phagocytes,  for  numerous  other  body  cells  show  this 
capacity,  including  the  eosinophiles,  the  endothelial  cells,  the  pulp  cells 
of  spleen  and  lymph-nodes,  connective  tissue  cells,  including  bone  cells, 
striated  muscle  cells  and  giant  cells.  It  is  probable  that  the  lymphocytes 
and  the  mast  cells  exhibit  no  phagocytic  activity.  Metchnikoff  divided 
phagocytes  into  microphages  and  macrophages.  The  microphages 
include  particularly  the  neutrophilic  leucocytes  and  the  eosinophilic 
leucocytes,  the  important  phagocytes  of  the  circulating  blood.  The 
macrophages  include  the  other  cells  mentioned  above,  the  most  important 
being  the  endothelial  cells.  It  is  perfectly  true  that  the  endothelial  cell 
circulates  in  the  blood,  but  apparently  its  most  important  activity  is  in 
the  tissues  and  body  spaces.  The  microphages  are  the  more  sensitive 
of  the  two  groups  and  react  not  only  to  chemical  stimuli  but  also  to 
tactile  and  physical  influences. 


154  THE  PRINCIPLES  OF  IMMUNOLOGY 

Functions  of  Phagocytosis. — Phagocytosis  plays  an  important  part 
in  the  entire  life  of  the  mammalia,  even  though  the  differentiation  of 
many  cells  excludes  them  from  this  function.  The  destruction  of 
erythrocytes  in  spleen,  liver  and  bone  marrow  is  in  part  due  to  phago- 
cytosis. Involution  of  the  uterus  after  pregnancy,  involution  of  senile 
ovaries,  decrease  in  substance  of  the  brain  and  other  organs  in  old  age 
are  due  to  phagocytosis.  Metchnikoff  has  laid  considerable  stress  upon 
the  activity  of  phagocytes  in  the  atrophic  processes  of  old  age.  Rind- 
fleisch  claims  to  have  demonstrated  that  phagocytes  are  active  in  the 
breaking  down  and  removal  of  gouty  deposits  in  and  about  joints.  The 
fixed  tissue  phagocytes  which  play  a  part  in  the  physiological  destruc- 
tion of  red  blood-corpuscles  have  been  designated  by  Kyes  as 
hemophages.  In  various  animal  species  the  blood  destruction  accom- 
plished by  the  hemophages  may  be  carried  on  predominately  in  one 
organ  or  another,  the  site  of  destruction,  however,  being  constant  for 
a  given  species  under  normal  conditions.  Pearce  and  his  co-workers 
have  shown  that  extensive  blood  destruction  increases  the  phagocytic 
activity  in  the  spleen  and  liver.  They  have  also  shown,  as  has  been 
confirmed  by  us,  that  the  removal  of  the  spleen  results  in  an  assump- 
tion of  hemophagocytic  activity  by  the  endothelial  cells  of  the  lymph- 
nodes.  Gary  has  demonstrated  that  the  injection  of  foreign  red 
blood-corpuscles  markedly  increases  the  hemophagocytic  activity  of  the 
recipient,  not  only  in  the  spleen,  which  normally  plays  the  important 
part  in  destruction  of  the  red  cells,  but  also  in  other  organs.  The 
resistance  of  the  organism  to  foreign  bodies,  either  living  or  inert, 
is  partly  the  result  of  the  same  process. 

Under  abnormal  circumstances  the  removal  of  tissue  and  cell 
detritus  is  due  in  part  to  phagocytosis.  In  the  inflammatory  reaction 
following  the  introduction  of  foreign  bodies,  especially  infective  bac- 
teria, phagocytosis  is  the  first  line  and  most  important  defensive 
mechanism  against  invasion.  Dusts  inhaled  into  the  lungs  are  taken 
up  by  mononuclear  phagocytes  or  macrophages  and  conveyed  to  neigh- 
boring lymphatics  and  lymph-nodes,  thus  preventing  accumulation  on 
the  respiratory  membrane.  In  inflammation  the  circulation  is  slowed 
in  the  small  vessels  of  the  neighborhood,  thus  permitting  the  accumu- 
lation of  leucocytes  on  the  inner  wall  of  the  vessels.  They  then  migrate 
through  the  vessel  walls  by  ameboid  movement  and  because  of  chemo- 
tactic  attraction  continue  through  the  tissues  to  the  irritating  sub- 
stances. If  the  latter  are  bacterial  the  leucocytes  attempt  to  ingest 
and  destroy  them.  Thus  it  can  be  seen  that  phagocytosis  is  an  im- 
portant process  in  the  normal  physiology  of  the  body  and  perhaps 
even  more  so  in  the  pathological  physiology  of  defense  against  disease. 

The  material  to  be  ingested  by  phagocytes  in  part  determines  the 
type  of  cell  which  participates.  The  microphages  are  especially  active 
in  taking  up  bacteria,  whereas  the  macrophages  are  active  in  ingesting 
inert  tissue  detritus.  Nevertheless,  macrophages  often  take  tip  bacteria, 
as  in  tuberculosis,  and,  as  has  been  shown  by  Kyes,  by  Bull  and  by 
Hopkins  and  Parker,  pneumococci,  typhoid  bacilli  and  streptococci  are 


FIG.   16. — Microscopic  drawing  showing  the  phagocytosis 

of   gonococci   by   the   polymorphonuclear   leucocytes   in 

urethral  pus. 


CELLULAR  RESISTANCE  155 

removed  from  the  circulation  by  endothelial  cells  lining  blood-vessels. 
Microphages  may  also  play  a  large  part  in  the  removal  of  tissue  detritus 
and  may  take  up  pigment  as  in  malaria. 

Experimental  Demonstration. — The  experimental  demonstration  of  phago- 
cytosis in  mammals  is  comparatively  simple,  as  the  following  experiment  from 
Metchnikoff  will  show.  The  blood  of  a  bird,  such  as  goose,  hen  or  pigeon,  is 
selected  because  of  the  fact  that  the  nucleated  erythrocytes  are  easily  distinguished 
from  those  of  mammals.  Defibrinated  bird  blood  mixed  with  equal  parts  salt 
solution  is  injected  (about  3.0  c.c.)  into  the  peritoneum  of  a  healthy  guinea-pig. 
Material  is  removed  for  study  by  means'  of  finely  drawn  out  glass  pipettes,  drops 
being  placed  on  slides  for  study  with  or  without  subsequent  staining.  Within 
the  first  hour  the  leucocytes  seem  to  have  disappeared  from  the  peritoneum. 

The  disappearance  is  particularly  striking  when  bacteria  are  injected  and 
was  interpreted  by  Metchnikoff  as  a  destruction  of  the  phagocytic  cells,  a 
phenomenon  which  he  called  phagolysis.  At  the  end  of  from  one  to  two  hours 
exudate  may  be  withdrawn  which  shows  numerous  cells,  particularly  macrophages. 
The  macrophages  show  ingestion  of  the  nucleated  erythrocytes  and  at  from 
twenty-four  to  forty-eight  hours  exhibit  digestive  vacuoles  and  partial  digestion 
of  the  erythrocytes. 

At  the  end  of  three  days  the  digestion  is  practically  complete. 
Metchnikoff  has  shown  that  immunization  will  definitely  limit  the  ap- 
pearance of  phagolysis.  Sanarelli,  however,  maintains  that  the  disap- 
pearance of  the  leucocytes  is  not  due  to  phagolysis  but  rather  to  the 
fact  that  the  leucocytes  of  the  peritoneal  cavity  and  of  the  blood 
accumulate  in  the  epiploic  appendages  into  which  the  bacteria  are 
likely  to  be  carried  by  the  lymphatic  stream.  Here,  he  asserts,  bac- 
teriolysis and  phagocytosis  progress  actively.  Hence  the  disappearance 
of  the  cells  from  the  exudate. 

A  similar  experiment  may  be  performed  with  a  suspension  of  pigment,  as 
for  example  5.0  c.c.  finely-divided  suspension  of  cinnabar  (red  mercuric  oxide). 
This  shows  no  digestion  but  active  phagocytosis  and  a  rapid  transfer  to  re- 
gional lymph-nodes. 

Phagocytosis  of  bacteria  may  be  very  well  demonstrated  with  colon  bacilli. 
It  is  desirable  in  this  instance  to  excite  some  exudation  before  the  introduction 
of  the  colon  bacilli.  This  may  be  produced  by  injecting  about  twelve  hours 
previously  10.0  c.c.  sterile  bouillon  or  aleuronat  suspension.  This  may  be  done 
in  the  evening  and  the  following  morning  the  guinea-pig  is  ready  for  the  injection 
of  a  24-hour  bouillon  culture  or  a  24-hour  slant  agar  culture  suspended  in  salt 
solution.  The  subsequent  phenomena  are  similar  to  those  following  the  injection 
of  bird  blood. 

The  Mechanism  of  Phagocytosis. — In  earlier  experiments  of  this 
sort  several  questions  as  to  the  mechanism  of  the  process  arose.  That 
the  bacteria  do  not  actively  penetrate  into  the  phagocytes  has  been 
demonstrated  by  direct  observation  of  the  ameboid  action  of  the  cells 
and  is  concluded  also  by  analogy  from  the  fact  that  non-motile  bacteria, 
non-motile  cells,  such  as  erythrocytes,  and  inert  bodies,  such  as  cinna- 
bar, are  readily  ingested  by  the  phagocytic  cells.  That  the  bacteria  are 
not  killed  before  ingestion  is  shown  by  the  fact  that  cultures  may  be 
successful  in  the  case  of  anthrax  bacilli  shortly  after  they  have  been 
taken  up  by  phagocytes.  This  may  also  be  illustrated  by  the  following 
experiment  with  the  use  of  neutral  red  as  a  vital  stain.  This  stains  only 
dead  cells  and  imparts  no  color  to  living  cells.  A  warm  hanging  drop 
preparation  of  the  exudate  from  a  guinea-pig  injected  with  colon 


156  THE  PRINCIPLES  OF  IMMUNOLOGY 

bacilli  as  outlined  above  may  be  mixed  with  a  drop  of  i  per  cent, 
neutral  red  in  isotonic  salt  solution.  At  first  the  extracellular  bacteria 
show  no  stain,  and  but  few  of  the  intracellular  bacilli  take  the  stain. 
As  time  goes  on  the  number  of  intracellular  organisms  taking  the  stain 
increases  until  they  are  completely  digested.  Metchnikoff  interpreted 
the  coloration  of  the  bacteria  as  being  due  to  an  acid  digesting  fluid 
formed  by  the  cell,  but  we  are  unable  to  state  at  the  present  time 
whether  the  digestion  of  the  bacteria  is  due  to  a  special  ferment  or 
due  to  the  same  ferments  that  digest  the  cells  themselves  after  they 
are  destroyed. 

The  Physical  Basis  of  Phagocytosis. — The  mechanism  of  phagocy- 
tosis both  as  regards  immunity  and  biology  in  general  has  been  the 
the  subject  of  much  investigation.  There  are  those  who  have  main- 
tained that  the  ameba  or  the  leucocyte,  in  spite  of  the  absence  of  a 
nervous  system,  exhibits  individual  intelligence  in  the  selection  of  the 
material  it  takes  up,  but  the  bulk  of  experimental  evidence  would  place 
the  phenomenon  largely  on  a  physical  chemical  basis.  There  are  im- 
portant differences  between  free  living  amebse  and  the  phagocytes  of 
higher  animal  life,  such  as  the  ectosarc  and  endosarc  of  the  amebse, 
its  pulsating  vacuoles,  variety  of  pseudopodia,  conjugation  and  cyst 
formation,  but  there  are  resemblances  in  movement,  form,  nutrition  and 
ultimate  genesis  which  form  a  basis  for  many  comparisons.  That  the 
life  activities  of  the  ameba  can  be  closely  simulated  by  non-living 
materials  has  been  known  for  many  years,  but  the  most  important 
stimulus  to  these  studies  in  recent  years  has  been  given  by  the  work 
of  Jennings.  A  fundamental  conception  necessary  to  understanding 
the  physical  basis  of  ameboid  motion  and  phagocytosis  is  that  of  the 
phenomena  of  surface  tension.  Wells  expresses  the  matter  most 
clearly  and  concisely  as  follows :  "  Imagine  a  drop  of  fluid  suspended 
in  water — let  it  be  a  drop  of  protoplasm,  or  oil,  or  mercury ;  the  drop 
owes  its  tendency  to  assume  a  spherical  shape  to  the  surface  tension, 
which  is  pulling  the  free  surface  toward  the  center  and  acting  with  the 
same  force  on  all  sides.  The  result  is  that  the  drop  is  surrounded  by 
what  amounts  to  an  elastic,  well-stretched  membrane,  similar  to  the 
condition  of  a  thin  rubber  bag  distended  with  fluid.  If  at  any  point 
in  the  surface  the  tension  is  lessened,  while  elsewhere  it  remains  the 
same,  of  necessity  the  wall  will  bulge  at  this  point,  the  contents  will  flow 
into  the  new  space  so  offered  and  the  rest  of  the  wall  will  contract; 
hence  the  drop  moves  toward  the  point  of  lowered  surface  tension. 
Conversely,  if  the  tension  is  increased  in  one  place  the  wall  at  this 
point  will  contract  with  greater  force  than  elsewhere,  driving  the  con- 
tents toward  the  less  resistant  part  of  the  surface,  and  the  drop  will 
move  away  from  the  point  of  increased  tension."  The  experimental 
demonstration  of  this  phenomenon  is  relatively  simple.  A  drop  of  mer- 
cury is  placed  in  a  nitric-acid  solution  and  near  it  is  placed  a  crystal  of 
potassium  dichromate.  A  yellow  color  diffuses  out  from  the  dichro- 
mate;  as  the  color  reaches  the  mercury  the  latter  begins  to  move 
toward  the  crystal.  This  is  the  result  of  oxidation  of  the  adjacent 


CELLULAR  RESISTANCE  157 

surface  of  the  mercury  drop  whereby  the  surface  tension  of  this  side 
is  lowered,  thus  causing  the  progressive  movement  in  the  direction  of 
the  dichromate  crystal.  Similarly  a  drop  of  clove  oil  in  a  mixture  of 
glycerol  and  alcohol  will  move  about  and  send  out  pseudopodia  in  much 
the  same  manner  as  an  ameba.  The  movement  depends  upon  the  solu- 
bility of  the  clove  oil  in  alcohol,  but  the  glycerin  retards  the  diffusion 
and  thus  determines  a  certain  degree  of  irregularity  in  the  movements. 
If  strong  alcohol  be  introduced  near  the  clove  oil  the  surface  tension 
of  the  oil  is  reduced  and  it  moves  toward  the  alcohol.  Heat  applied 
near  one  side  of  the  drop  will  also  lower  the  surface  tension,  and  it 
moves  toward  the  point  of  heat — positive  thermotaxis.  These  experi- 
ments illustrate  the  physical  basis  of  ameboid  movement,  but  do  not 
explain  ingestion  of  particles.  For  this  purpose  a  drop  of  chloroform 
may  be  placed  in  water  and  brought  near  a  variety  of  objects,  such  as 
glass  particles  and  small  pieces  of  shellac,  paraffin  and  glass.  Such  a 
drop  will  flow  around  a  piece  of  shellac  and  dissolve  it.  A  piece  of 
glass  covered  with  shellac  will  be  taken  up,  the  shellac  dissolved  and 
the  piece  of  glass  then  extruded.  If  a  long  "  hair "  of  shellac  is 
brought  into  contact  with  the  chloroform,  the  former  will  be  bent  in 
the  middle,  pseudopodia  will  extend  along  it  and  it  will  finally  be 
curled  up  inside  the  drop  and  dissolved.  These  various  activities  of 
the  oil  drop  or  chloroform  drop  resemble  in  detail  the  activities  of 
amebse  under  similar  circumstances  and  may  be  understood  as  indi- 
cating that  the  process  of  phagocytosis  is  based  on  definite  physical 
laws.  The  experiments  do  not  explain  all  the  phenomena,  however, 
and  must  be  interpreted  as  solving  the  problem  only  partially.  Various 
food  particles  are  not  soluble  in  the  cytoplasm  of  the  ameba,  bacteria 
are  not  soluble  in  the  cytoplasm  of  the  leucocytes,  but  in  each  instance 
must  be  digested  in  some  way.  Furthermore,  the  phagocytes  have  the 
property  of  taking  up  inert  and  insoluble  particles  such  as  coal  dust 
and  other  pigments,  substances  which  cannot  exert  chemotaxis  nor 
alter  surface  tension.  The  artificial  ameba  does  not  assimilate,  it 
merely  dissolves.  An  additional  differentiation  between  the  leucocytes 
and  the  ameba  is  the  fact  that  the  ameba  is  a  free  living  organism 
capable  of  nourishing  itself  independently  of  life  within  another  or- 
ganism. On  the  other  hand,  the  leucocyte  depends  upon  the  blood  for 
its  nutrition  and  differs  in  ameboid  movement  and  irritability  from 
the  free  living  ameba.  Thus  we  must  conclude  that  the  problem  of 
phagocytosis  is  not  solved  by  these  experiments  and  that  the  life  activ- 
ities of  these  cells  are  not  as  yet  explainable  on  a  purely  physical  basis. 
Influence  of  Temperature  on  Phagocytosis. — Madsen  and  his 
school  have  made  accurate  studies  of  the  influence  of  temperature 
on  phagocytosis.  They  have  shown  that  within  certain  limits  the 
phenomenon  of  phagocytosis  increases  with  the  degree  of  tem- 
perature. Starting  at  a  point  of  =±=  5°  C,  the  phagocytic  power 
increases  with  temperature  up  to  the  normal  temperature  of  the  species 
from  which  the  phagocytic  cells  are  derived.  In  cold-blooded  animals, 
on  the  other  hand,  the  temperature  of  the  environment  within  certain 


158  THE  PRINCIPLES  OF  IMMUNOLOGY 

limits  appears  to  have  no  influence  whatever  on  the  phagocytic  activity 
of  their  cells.  Calderone  and  Runfola  have  recently  studied  the  in- 
fluence of  temperature  upon  phagocytosis  in  the  frog  and  find  that 
phagocytosis  proceeds  actively  between  5°  and  40°  C,  but  ceases  when 
a  temperature  of  42°  C.  is  reached. 

OPSONINS 

Introduction. — Early  in  the  study  of  phagocytosis  it  was  noted 
that  immune  animals  respond  to  the  introduction  of  the  antigenic 
bacteria  by  a  greater  degree  of  phagocytic  activity  than  normal  animals. 
This  was  interpreted  by  Metchnikoff  as  being  due  to  "  stimulins " 
which  were  supposed  to  augment  the  activity  of  the  phagocytic  cells. 
The  first  study  of  importance  in  contraindication  of  MetchnikorFs  con- 
ception of  stimulins  was  that  of  Denys  and  Leclef  in  1895.  They 
showed  in  a  study  of  streptococcus  immunity  in  rabbits  that  the  leuco- 
cytes of  normal  and  immune  animals  took  up  the  bacteria  equally 
well,  but  that  both  varieties  of  leucocytes  acted  much  more  powerfully 
when  immune  serum  was  added.  They  indicated  that  the  process  of 
immunization  did  not  augment  the  phagocytic  power  of  the  leucocytes 
and  concluded  that  in  their  opinion  the  antitoxic  substance  acts  not 
upon  the  leucocyte  but  upon  a  poison  enclosed  within  the  bodies  of  the 
microbes  or  dissolved  in  the  medium,  the  poison  acting  to  protect  the 
bacteria  against  the  attacks  of  the  leucocyte  until  neutralized  by  the 
immune  substance  in  the  serum.  The  observations  were  confirmed  by 
other  investigators  and  later  Denys  and  Leclef  showed  that  whereas 
extremely  virulent  bacteria  are  taken  up  by  leucocytes  in  normal  serum 
to  only  a  slight  degree,  the  addition  of  an  immune  serum  markedly 
increases  the  phagocytosis.  Little  progress  was  made  until  after  the 
discovery  by  Leishman  whereby  the  study  of  phagocytosis  could  be 
carried  out  in  vitro.  Modifying  this  method,  Wright  and  Douglas  in 
1903  published  the  first  of  a  series  of  experiments  which  have  built  up 
in  large  measure  our  modern  conception  of  the  influence  of  serum  on 
phagocytosis  and  the  practical  use  of  bacterial  vaccination  in  the  treat- 
ment of  disease.  They  showed  conclusively  that  it  is  the  activity  upon 
the  bacteria  of  some  substance  in  the  blood  which  favors  phago- 
cytosis and  they  named  the  substance  opsonin.  By  treating  the  bacteria 
with  serum,  then  washing  them  to  remove  the  serum,  from  the  sur- 
rounding medium  and  finally  mixing  with  a  leucocyte  emulsion  in  salt 
solution  they  showed  that  phagocytosis  proceeds  actively.  Similar 
treatment  of  the  leucocytes  by  serum  produces  no  augmentation  of  their 
phagocytic  activity.  Thus  it  was  shown  that  the  serum  does  not  stimu- 
late the  leucocytes  but  rather  prepares  the  bacteria  so  that  they  may 
more  readily  be  ingested,  hence  the  term  opsonin  (Gr.  opsono — to  pre- 
pare food).  There  is  some  variation,  however,  in  the  way  the  serum 
operates  in  the  case  of  different  bacteria.  Tunnicliff  and  Davis  have 
shown  that  fusiform  bacilli  and  influenza  bacilli  can  be  taken  up  readily 
independently  of  the  presence  of  serum.  There  are,  of  course,  different 
degrees  of  facility  with  which  bacteria  can  be  taken  up,  varying  from 


CELLULAR  RESISTANCE  159 

those  which  absolutely  require  the  intervention  of  an  opsonin 
and  those  mentioned  above,  which  apparently  need  little  or  no  par- 
ticular opsonization. 

Experimental  Demonstration. — For  the  experimental  demonstration,  of 
opsonization  it  is  necessary  to  have  washed  leucocytes,  bacterial  suspension  and 
blood  serum.  Large  quantities  of  leucocytes  may  be  obtained  by  injecting  5.0 
c.c.  aleuronat  suspension  into  a  guinea-pig's  peritoneum  and  withdrawing  the 
exudate  at  the  end  of  twelve  to  twenty-four  hours.  These  may  be  suspended 
in  five  to  ten  volumes  normal  saline,  gently  mixed  and  centrifuged,  the  process 
being  carried  out  three  times,  when  the  cells  are  said  to  have  been  washed  three 
times.  If  human  leucocytes  are  desired,  10.0  c.c.  saline  sodium  citrate  are  placed 
in  a  centrifuge  tube  and  2.0  c.c.  blood  added.  The  tube  is  centrifuged  at  high 
speed,  whereupon  a  layer  or  "  cream  "  of  leucocytes  collects  at  the  upper  level 
of  the  cell  mass.  The  cream  can  be  removed  by  a  drawn-out  nipple  pipette 
and  the  cells  washed  as  indicated  for  the  peritoneal  exudate.  The  bacterial 
emulsion  can  be  made  from  a  twenty-four-hour  slant  agar  culture  of  staphylo- 
coccus  pyogenes  aureus  by  adding  10.0  c.c.  salt  solution,  allowing  to  stand  for 
ten  to  fifteen  minutes  and  then  rotating  the  tube  between  the  palms  of  the  hands. 
This  is  pipetted  into  another  tube  and  for  safety  may  be  killed  by  heating  in  a 
water  bath  at  55°-6o°  C.  for  two  hours.  The  serum  may  be  obtained  by  allowing 
blood  to  clot  and  then  drawing  off  the  serum.  Small  quantities  may  be  obtained 
by  the  use  of  a  tube  such  as  shown  in  Fig.  8.  Having  these  ready,  0.5  c.c. 
bacterial  emulsion  are  mixed  with  o.i  c.c.  serum  and  incubated  for  one-half 
hour,  then  washed  three  times  and  the  organisms  resuspended  in  0.5  c.c.  saline. 
Several  capillary  pipettes  are  made  from  glass  tubing  (5  mm.  bore)  and  the 
upper  end  flanged  so  as  to  take  a  rubber  nipple.  A  mark  is  made  with  a  grease 
pencil  about  2  cm.  from  the  tip,  which  serves  as  a  volume  indicator.  (Fig.  n.)  In 
the  experiment  one  volume  bacterial  suspension,  one  volume  bacterial  emulsion  and 
one  volume  serum  or  saline  are  drawn  into  the  pipette  each  in  succession  to 
the  mark,  permitting  a  small  amount  of  air  to  enter  before  the  next  volume  is 
taken  up  so  as  to  permit  of  exact  measurement  of  the  volume.  These  are  blown 
into  a  watch  crystal  and  mixed  by  blowing  in  and  out  several  times ;  then  taken 
into  the  capillary  again  and  the  end  sealed.  After  incubation  at  37°  C.  for 
fifteen  minutes  the  tip  is  broken,  the  mixture  dropped  on  slides  or  cover  slips, 
spread,  dried  and  stained  with  Wright's  stain  or  some  other  modification  of 
the  Romanowsky  stain.  Then  the  number  of  bacteria  in  a  given  number  of 
leucocytes  (20  to  50  or  more)  are  counted  and  the  average  calculated.  A  sample 
protocol  follows : 

Average 

phagocytosis 

per  cell 

1.  Leucocytes  (washed)  -f  bacteria  (untreated)  -f  serum  22 

2.  Leucocytes  (washed)  -J-  bacteria  (untreated)  -j-  NaCl    I 

3.  Leucocytes  (washed)  -j-  bacteria  (treated)       -j-  NaCl    14 

In  the  above  protocol  it  is  seen  that  the  leucocytes  exhibit  a  slight  capacity 
for  taking  up  bacteria  independently  of  the  presence  of  serum,  but  that  this  is 
much  augmented  either  in  the  presence  of  serum  or  by  previously  treating  the 
bacteria  with  serum. 

Normal  Opsonins. — As  has  been  indicated,  the  phagocytosis  of  bac- 
teria and  other  cells  is  greater  in  immune  than  in  normal  animals,  the 
difference  being  due  to  increase  in  the  opsonin  content  of  the  serum 
of  the  immune  animal.  It  was  soon  observed  that  the  opsonin  of  the 
serum  of  normal  animals  could  be  destroyed  by  heat  to  60°  to  65°  C. 
for  10  to  15  minutes;  whereas  the  opsonin  of  immune  animals  is  not 
destroyed  by  heat  of  62°  to  63°  C.  for  forty-five  minutes.  Similarly 
exposure  to  light  at  room  temperature  leads  to  deterioration  of  normal 
opsonin  in  a  few  days  but  has  practically  no  effect  on  immune  opsonin. 
These  differences  in  behavior  were  at  first  thought  to  constitute  an 


160  THE  PRINCIPLES  OF  IMMUNOLOGY 

actual  difference  in  the  nature  of  normal  and  immune  opsonin,  but 
this  view  has  now  been  almost  entirely  abandoned.  In  the  discussion 
of  this  change  of  view  it  is  essential  to  present  first  the  development  of 
work  in  regard  to  the  normal  opsonin.  Conservative  workers  were 
not  disposed  to  accept  the  opsonin  as  a  new  form  of  antibody  and 
from  the  ease  of  deterioration  of  the  normal  opsonin  thought  that  it 
was  identical  with  complement.  Furthermore,  it  was  shown  that  fixa- 
tion of  complement  by  a  hemolytic  system  or  sensitized  bacteria  re- 
moves the  opsonin,  that  yeast  cells,  cell  detritus  and  bacteria  will  absorb 
both  opsonin  and  complement,  that  blood  serum  and  edema  fluids 
contain  parallel  amounts  of  opsonin  and  complement,  that  certain  body 
fluids,  such  as  the  aqueous  humor  of  the  eye,  contain  neither  complement 
nor  opsonin.  Nevertheless,  the  removal  of  complement,  as  by  heating, 
does  not,  as  Hektoen  has  shown,  remove  all  the  normal  opsonic  power 
of  the  serum;  and  the  fixation  of  complement  by  a  hemolytic  system 
or  by  sensitized  bacteria  still  leaves  slight  opsonic  power  in  the  serum. 
The  addition  of  fresh  serum  to  a  slightly  active  heated  serum  restores 
the  activity  practically  to  normal  in  much  the  same  manner  as  a 
hemolytic  amboceptor  may  be  reactivated  by  complement.  The  fol- 
lowing example  taken  from  Cowie  and  Chapman  and  slightly  modified 
serves  to  illustrate  this  reactivation.  The  substances  indicated  in  the 
protocol  are  added  to  leucocyte  and  bacterial  emulsions  and  the  figures 
given  are  for  the  bacterial  count  per  leucocyte: 

1.  Unheated  (normal)  serum  1544 

2.  Salt  solution 0.18 

3.  Heated  serum  57°  C 1.08 

4.  Normal  unheated  serum,  diluted  i  to  15  1.56 

5.  Heated  serum  -f-  normal  serum  diluted  i  to  15 12.40 

6.  Two  volumes  unheated  normal  serum 16.08 

Thus  it  will  be  seen  that  heating  the  serum  reduces  the  phagocytic 
index  from  15.44  to  1.08;  that  normal  serum,  diluted  so  that  its 
phagocytic  index  is  reduced  to  1.56,  added  to  heated  serum,  raises 
the  index  to  12.40,  much  higher  than  can  be  accounted  for  by  the 
total  indices  of  the  two  components.  It  can  then  be  concluded  that 
the  normal  opsonic  power  of  serum  depends  upon  two  factors,  a  weakly 
acting  thermostable  element  and  a  thermolabile  element  which  markedly 
adds  to  the  combined  power  of  the  mixture.  Cowie  and  Chapman 
have  shown  that  at  o°  C.  the  thermostable  element  of  opsonin  is  ab- 
sorbed by  the  bacteria,  but  that  the  thermolabile  element  remains  in 
the  supernatant  fluid  and  is  capable  of  reactivating  a  heated  serum. 
It  has  also  been  demonstrated  by  absorption  experiments  that  the  ther- 
mostable element  is  specific.  Numerous  continental  workers  contradict 
this  statement,  but  their  studies  have,  for  the  most  part,  ignored  the 
existence  of  the  thermostable  element  of  normal  opsonins.  Hektoen 
has  shown  that  saturation  of  the  bacteria  with  opsonin  and  heating  so 
as  to  destroy  the  thermolabile  part  leaves  the  bacteria  in  such  condi- 
tion that  they  cannot  absorb  any  more  opsonin  from  another  serum. 


CELLULAR  RESISTANCE  161 

Moore  has  found  that  in  guinea-pigs  "  the  complement  titer  varies 
with  the  opsonic  index  and  in  the  same  direction."  These  facts,  to- 
gether with  the  fact  that  vaccination  with  bacteria  will  increase  specifi- 
cally the  opsonic  content  of  the  blood  suggest  a  close  similarity  of 
opsonins  to  agglutinins  and  amboceptors.  The  resemblance  to  agglu- 
tinins  is  only  relative  for  as  we  have  seen  the  thermostable  element  of 
opsonin  is  markedly  augmented  in  activity  by  the  addition  of  fresh 
serum,  whereas  agglutinins  are  not  affected  in  any  way  by  the  addition 
of  complementary  substance.  Hektoen  has  shown  that  in  the  process 
of  immunization  the  curves  of  opsonin  and  agglutinin  production  are 
nearly  parallel,  but  that  heating  does  not  influence  the  agglutinin  and 
markedly  depresses  the  opsonic  action,  the  latter  being  restored  by  the 
addition  of  fresh  normal  serum.  Levaditi,  in  a  study  of  the  site  of 
formation  of  opsonins,  showed  that  certain  organs  rich  in  agglutinin 
contain  no  opsonins.  The  thermostability  and  specific  absorption  of 
opsonins  suggest  similarity  to  amboceptors,  but  the  amboceptors  are 
not  capable  of  acting  without  complement  whilst  the  opsonin  is  capable 
of  acting  independently  of  fresh  serum.  The  fresh  serum  augments 
the  activity  of  the  thermostable  element  of  opsonins  but  is  not  an 
absolute  essential  for  activity.  That  the  opsonin  is  not  identical 
with  hemolytic  and  bactericidal  amboceptors  is  indicated  by  the  fact 
that  there  are  such  amboceptors  in  sera  which  have  no  opsonic  power  ; 
that  in  sera  which  show  both  amboceptors  and  opsonins  there  is  no 
parallelism  between  the  activity  of  the  two.  Sera  may  be  strongly 
opsonic  for  certain  bacteria  and  yet  contain  no  bactericidal  amboceptor. 
Much  of  the  material  quoted  above  has  been  worked  out  iri  connection 
with  immune  opsonins,  but  nevertheless  it  is  safe  to  conclude  that  the 
opsonic  action  of  normal  serum  depends  upon  the  operation  of  two 
elements,  a  thermostable  element  which  behaves  as  a  "  facultative " 
amboceptor  and  a  thermolabile  element  which,  if  not  identical  with, 
resembles  complement  most  closely. 

Immune  Opsonins. — As  has  been  indicated,  it  is  possible  by  im- 
munization to  increase  to  a  very  considerable  degree  the  opsonic  activity 
of  serum.  The  immune  opsonins  were  considered  as  of  a  constitution 
different  from  the  normal  opsonin  because  of  the  claim  that  the  appli- 
cation of  heat  did  not  alter  their  activity.  Dean  showed,  however,  that 
this  assumption  is  not  true  for  he  found  that  heating  to  60°  C.  definitely 
though  not  very  markedly  reduces  the  opsonic  activity  of  immune  serum, 
and  that  reactivation  takes  place  on  the  addition  of  a  fresh  normal 
serum.  The  following  protocol  shows  the  phagocytic  index  as  deter- 
mined by  the  use  of  various  sera  and  mixtures : 

Normal  serum   1 1.9 

Heated  immune  serum 7.1 

Heated  immune  serum  +  normal  serum 33.0 

Hektoen  reached  the  same  conclusion  with  the  hemopsonic  power  of 
rabbits  immunized  to  goat  erythrocytes,  diluting  the  serum  so  that  it 
ii 


1 62  THE  PRINCIPLES  OF  IMMUNOLOGY 

showed  minimal  opsonic  power  and  no  hemolytic  action.  One  protocol 
from  his  work  serves  to  illustrate. 

Heated  immune  serum          Fresh  guinea-pig  serum  Phagocytosis 

o.ooi  4 

0.001  0.01  20 
O.OI  O 

Levaditi  and  Koessler  showed  that  a  serum  which  contained  anti- 
complement  by  virtue  of  immunization  with  complement,  when  added  to 
an  immune  opsonin,  noticeably  reduces  the  opsonic  power. 

The  full  activity  of  the  immune  opsonin  depends,  as  can  be  seen, 
from  the  above  experiments,  upon  a  thermostable  and  a  thermolabile 
element,  as  is  true  of  the  normal  opsonin,  but  the  activation  by  fresh 
serum  in  case  of  the  thermostable  element  of  immune  opsonin  is  pro- 
portionately much  less  than  activation  of  thermostable  normal  opsonin 
by  fresh  serum.  Reference  to  the  activation  of  a  hemolytic  amboceptor 
by  complement  shows  that  a  given  amount  of  complement  will  activate 
a  very  small  amount  of  amboceptor  in  greater  proportion  than  a  large 
amount  of  amboceptor.  The  thermostable  fraction  of  opsonin  has 
been  referred  to  as  a  facultative  amboceptor,  because  the  action  of  the 
thermolabile  part  is  not  essential.  Assuming  this  interpretation  to  be 
correct  and  assuming  that  the  thermolabile  element  operates  as  a  com- 
plement, it  is  a  simple  matter  to  infer  that  this  complement  would  have 
a  proportionately  larger  action  on  the  facultative  amboceptor  of  normal 
opsonin,  which  is  present  in  very  small  amount,  than  on  the  similar  am- 
boceptor of  immune  opsonin,  which  is  present  in  relatively  large  amount. 

Bacteriotropins. — Neuf  eld  and  his  school  maintain  that  the  immune 
opsonin  is  a  body  which  operates  only  in  the  presence  of  complement 
and  that  the  tropins,  bacteriotropins  and  cytotropins  are  bodies  appear- 
ing in  serum  which  has  been  rendered  complement-free,  and  which 
exhibit  a  capacity  for  so  altering  bacteria  or  cells  that  they  are  easily 
taken  up  by  phagocytes.  Levaditi  and  numerous  other  authors  agree 
that  Neufeld  has  shown  that  the  tropins  are  not  identical  with  those 
amboceptors  which  lead  to  cytolysis,  but  also  agree  that  Neufeld  has 
not  succeeded  in  demonstrating  that  the  tropins  are  antibodies  distinct 
and  apart  from  the  thermostable  element  of  immune  opsonin. 

Opsonins  for  Cells  other  than  Bacteria. — Numerous  substances, 
including  vegetable  cells,  such  as  yeasts,  and  bacteria,  as  well  as  a 
variety  of  animal  cells,  may  undergo  phagocytosis  when  influenced  by 
opsonins.  In  connection  with  phagocytosis  of  animal  cells  the  work  of 
Hektoen  and  his  collaborators  has  been  most  extensive.  The  investiga- 
tions have  thrown  much  light  on  the  general  study  of  opsonins  and,  di- 
rected particularly  toward  erythrocytes,  have  shown  that  the  same 
general  laws  governing  the  phagocytosis  of  bacteria  operate  in  the 
phagocytosis  of  erythrocytes.  Neufeld  and  Handel  have  shown  that 
emulsions  of  fat  droplets  in  protehvcontaining  media  can  serve  as 
excitants  of  the  formation  of  specific  opsonic  sera  but  conclude  that  in 
these  instances  the  protein  capsule  of  the  fat  droplets  which  serves  to 
stabilize  the  emulsion  is  the  important  factor  in  the  phenomenon.  Led- 


CELLULAR  RESISTANCE  163 

ingham  has  also  shown  that  the  injection  of  melanin  produces  a  specific 
opsonic  serum  and  others  have  shown  that  carbon  granules,  cinnabar, 
carmine,  etc.,  are  phagocyted  much  more  readily  in  the  presence  of 
serum  than  otherwise.  In  these  latter  instances  it  seems  probable  that 
the  serum  provides  a  protein  capsule  for  the  pigment  granules,  thus 
facilitating  the  action  of  opsonin,  but  at  the  present  time  no  satisfactory 
explanation  has  been  offered  for  the  production  of  a  specific  immune 
opsonin  following  the  injection  of  melanin.  Neufeld  and  Ungermann 
point  out  the  difficulty  of  satisfactory  measurement  of  phagocytic  action 
against  pigment  granules,  and  it  is  possible  that  this  source  of  error 
may  be  sufficient  to  throw  doubt  on  the  results  claimed  to  have  been 
obtained  with  insoluble  pigments. 

Specificity  and  other  Characters  of  Opsonins. — The  specificity  of 
the  immune  opsonins  is  clear-cut,  as  has  been  shown  by  numerous  in- 
vestigators. An  immune  opsonin  produced  by  vaccination  with  staphyl- 
ococci  shows  a  marked  influence  on  the  phagocytosis  of  the  antigenic 
organisms  but  none  whatever  on  non-related  organisms  such  as  colon 
bacilli.  As  in  the  case  of  other  immune  bodies,  group  reactions  are 
demonstrable.  Vaccination  with  typhoid  bacilli  leads  to  the  formation 
of  immune  opsonins  which  operate  in  high  degree  on  the  antigenic 
organism  and  also  to  less  degree  on  closely-related  organisms  such  as 
those  of  the  paratyphoid  groups.  Dean,  in  working  with  serum  dilu- 
tions in  order  to  demonstrate  that  an  optimum  concentration  of  opsonin 
may  not  necessarily  be  found  in  undiluted  serum,  reports  the  following 
experiment.  This  may  be  interpreted  as  showing  an  inhibition  zone  in 
the  stronger  concentrations,  although  the  differences  are  so  slight  as 
to  fall  within  the  limit  of  experimental  error. 

Dilution  of  serum  Phagocytic  index* 

o  97 

1-2  9.6 

1-4  i  o.o 

1-8  8.2 

1-16  8.5 

1-32  6.4 


*  Average  number  of  bacteria  ingested  per  leucocyte. 

Influence  of  Phagocyte  and  Ingested  Elements. — The  foregoing 
paragraphs  have  considered  the  influence  of  serum  on  phagocytosis,  but 
detailed  studies  have  shown  that  certain  considerations  in  regard  to  both 
the  bacteria  and  the  leucocytes  exercise  some  influence.  Neufeld  pointed 
out  that  bacterial  cultures  from  ten  to  twenty-four  hours  old  are  best 
for  in  vitro  experiments.  The  reaction  takes  place  best  when  the  bac- 
teria are  suspended  in  equal-parts  broth  and  physiological  salt  solution, 
but  in  ordinary  laboratory  practice  salt  solution  is  used  without  the 
addition  of  broth  and  whatever  deterring  action  is  exercised  by  the  salt 
is  constant  in  the  series  of  experiments.  The  thickness  of  the  sus- 
pension is  of  importance  since  very  thin  suspensions  determine  a 
reduction  in  phagocytic  index  as  compared  with  thicker  suspensions. 
The  optimal  density  of  the  suspensions  varies  with  different  bacteria 


164  THE  PRINCIPLES  OF  IMMUNOLOGY 

and  must  be  determined,  in  exact  work,  for  the  organisms  under  investi- 
gation. The  more  homogeneous  the  emulsion,  the  better  the  phago- 
cytosis observed.  Numerous  investigators  have  shown  that  under 
experimental  conditions,  bacteria  killed  by  chemicals  or  by  heat  are 
phagocyted  at  precisely  the  same  rate  as  living  organisms.  Further- 
more, the  previous  staining  of  the  organisms  has  no  deterrent  action 
on  phagocytosis. 

Relation  of  Bacterial  Virulence. — The  relation  of  bacterial  viru- 
lence to  phagocytosis  has  been  the  subject  of  much  research  since 
Marchand  first  showed  that  virulent  streptococci  are  taken  up  hardly 
at  all  under  conditions  where  avirulent  streptococci  are  phagocyted 
with  avidity.  He  demonstrated  that  this  difference  is  not  due  to  the 
vitality  of  the  bacteria,  for  when  killed  by  heat  at  60°  C,  1.8  per  cent. 
HC1,  2.5  per  cent.  Na2CO3  or  90  per  cent,  alcohol,  the  virulent  forms 
show  the  same  resistance  to  phagocytosis.  Wright  and  also  Levaditi 
showed  that  the  same  difference  is  observable  in  the  case  of  phago- 
cytosis, without  the  intervention  of  opsonins.  Rosenow  confirmed 
Marchand's  results  by  the  use  of  freshly-isolated  virulent  pneumococci. 
Reduction  of  virulence  of  thirty-six  strains  by  repeated  cultivation  on 
media  resulted  in  increased  susceptibility  to  phagocytosis;  and  a  res- 
toration of  virulence  by  animal  passages  led  again  to  decreased  phago- 
cytosis. There  is,  however,  no  absolute  parallelism  between  virulence 
and  susceptibility  to  phagocytosis.  Markl,  von  Gruber  and  Futaki,  as 
well  as  Lohlein  and  others,  found  that  anthrax  bacilli  and  plague  bacilli 
when  taken  from  culture  material  are  actively  phagocyted  in  vitro  even 
though  highly  virulent  for  animals.  If  removed  from  a  guinea-pig's 
peritoneum  after  having  grown  there  for  several  hours,  they  are  no 
longer  phagocyted  in  vitro.  In  animal  experiments  they  are  at  first  the 
victims  of  active  phagocytosis  in  vivo,  but  after  several  hours  are  re- 
sistant to  phagocytosis.  Proper  staining  shows  that  in  the  resistant 
stage  the  organisms  show  definite  capsule  formation.  These  experi- 
ments indicate  that  the  resistance  is  entirely  a  function  of  the  bacteria, 
but  that  there  is  some  interdependence  between  the  bacteria  and  the 
opsonin  is  indicated  by  the  experiments  of  Ungermann,  who  worked 
with  pneumococci  virulent  for  mice  in  doses  as  small  as  0.000,001  c.c., 
but  not  injurious  for  rabbits  in  doses  as  large  as  i.o  c.c.  He  found 
that  mouse  serum  has  no  opsonic  action  and  that  rabbit  serum  acts 
energetically.  After  repeated  cultivation  so  as  to  reduce  virulence  for 
mice  the  organisms  are  opsonized  by  mouse  serum.  Von  Bockstaele 
and  also  Denys  and  von  den  Bergh  were  able  to  see  leucocytes  in  the 
presence  of  a  normal  serum  approach  and  even  break  up  chains  of  viru- 
lent streptococci  without  engulfing  them;  if  a  strong  immune  serum 
were  added,  there  resulted  active  phagocytosis.  In  summary,  these 
various  experiments  show  that  the  possession  of  virulence  by  an  organ- 
ism confers  upon  it  the  power  of  resisting  opsonization,  that  this  power 
has  some  relation  to  the  susceptibility  of  the  particular  animal  whose 
serum  is  used  for  opsonization,  that  the  resistance  to  opsonization  is 
not  lost  on  the  death  of  the  bacteria,  and  that  in  certain  instances  this 


CELLULAR  RESISTANCE  165 

resistance  is  accompanied  by  capsule  formation.  Levaditi  believes  that 
the  resistance  of  virulent  bacteria  is  dependent  upon  some  alteration 
of  the  bacterial  membrane  (which  alteration  determines  in  all  prob- 
ability the  virulence  of  the  organism)  and  also  perhaps  on  the  formation 
by  the  bacteria  of  an  anti-opsonic  or  anti-phagocytic  substance.  In  the 
latter  connection  Tschistowitsch  and  Jurewitsch  claim  to  have  shown 
that  on  washing,  virulent  pneumococci  lose  their  resistance  to  phago- 
cytosis, but  that  submitting  the  organisms  to  the  action  of  the  material 
in  the  washings  restores  them  again  to  their  resistant  state.  They  con- 
sidered that  the  salt  solution  removed  in  the  washing  a  secretion  which 
they  called  antiphagin.  This  work  has  not  been  confirmed  and  can- 
not be  regarded  as  establishing  beyond  question  the  existence  of 
an  antiphagin. 

Influences  Operating  upon  Phagocytic  Cells. — In  the  preliminary 
paragraphs  of  this  discussion  the  stimulin  theory  of  Metchnikoff  was 
dismissed  with  a  simple  statement  that  such  a  theory  exists.  Never- 
theless, the  leucocytes  and  their  possible  alterations  are  of  considerable 
importance  in  phagocytosis,  and  while  it  is  true  that  increased  phago- 
cytosis resulting  from  immunity  is  not  the  result  of  stimulins,  neverthe- 
less, it  is  possible  to  augment  the  activity  of  these  cells.  Neisser  and 
Guerrini  gave  the  name  Icuco  stimulants  to  certain  substances  which 
directly  act  upon  the  leucocytes.  According  to  Manwaring  and  Ruh, 
numerous  antiseptics  in  proper  concentration  exhibit  a  stimulating 
action.  According  to  others,  calcium  chloride,  magnesium  salts, 
potassium  iodide,  iodoform,  fat  soluble  substances  (except  cholesterol), 
substances  facilitating  oxidation,  pepton,  quinine  in  certain  low  con- 
centrations, nucleinic  acid,  similarly  excite  increased  phagocytosis. 
Marbe  has  extracted  a  thermostable  body  from  the  thyroid  gland  which 
excites  phagocytosis.  The  demonstration  that  the  action  of  these  various 
substances  is  upon  the  leucocytes  depends  upon  the  use  of  decreasing 
dilutions  of  the  substances  in  the  presence  of  sensitized  bacteria  and 
washed  leucocytes. 

Metchnikoff  showed  the  influence  of  heat  on  the  leucocytes  in 
experiments  which  are  tabulated  as  follows : 

Degree  of  heat  Time  of  heating  Phagocytic  index 

40°  C.  15  minutes  18 

45°  C.  15  minutes  8 

50°  C.  15  minutes  3 

55°  C.  5  minutes  1.2 

60°  C.  5  minutes  o 

60°  C.  30  minutes  o 

In  addition  to  heat,  alterations  of  OH  ions,  alterations  of  osmotic 
pressure,  cholesterol,  reduction  in  amount  of  electrolytes,  potassium 
ions,  alcohols,  ether,  quinine  and  certain  other  of  the  leucostimulants 
in  high  concentrations  act  upon  the  leucocytes  to  depress  their  phago- 
cytic  activity. 

Analysis  of  Mechanism  of  Phagocytosis. — The  mechanism  of 
phagocytosis  includes  the  approach  of  phagocytes  and  the  object  to  be 


1 66  THE  PRINCIPLES  OF  IMMUNOLOGY 

phagocyted,  the  ingestion  of  these  objects  and  in  the  case  of  living 
objects  their  death ;  finally  the  digestion  of  bacteria  and  other  suitable 
objects.  The  approach  of  the  cells  and  the  phagocytable  objects  is, 
according  to  Mesnil  and  his  co-workers  and  also  Levaditi,  due  to  a 
physical  chemical  reaction  and  not  dependent  on  the  life  of  the  leu- 
cocyte. If  leucocytes  are  injured  by  heat  to  45°,  50°  or  60°  C,  by  refrig- 
erator temperature,  by  shaking,  by  grinding,  and  then  mixed  with  bacteria 
and  inactivated  immune  serum,  the  bacteria  become  clumped  about 
the  leucocytes.  This  reaction  may  be  observed  even  if  the  tubes  are 
laid  in  melting  ice.  The  leucocytes  that  have  been  killed  or  paralyzed 
will  not  ingest  the  bacteria.  The  "  anchoring "  of  leucocytes  and 
bacteria  will  not  occur  unless  specific  opsonin  is  present  in  the  serum. 
It  occurs  with  fragments  of  leucocytes  as  well  as  other  cells  of  the 
leucocyte  series,  such  as  myelocytes  and  myeloblasts.  Thus  the  affinity 
may  be  expressed  as  existing  between  the  protoplasm  of  the  phagocytic 
cell  and  the  sensitized  bacteria  or  other  phagocytable  object. 

Although  actual  ingestion  of  objects  may  be  shown  in  the  case  of 
artificial  amebse  it  does  not  occur  in  the  leucocyte  unless  the  cell  is 
alive  and  in  possession  of  its  capacity  to  project  pseudopodia.  Hence 
this  stage  of  phagocytosis  must  be  bound  up  with  the  life  processes  of 
the  phagocyte. 

From  the  earlier  studies  of  Metchnikoff  it  has  been  known  that  the 
bacteria,  after  phagocytosis,  are  killed  and  digested.  The  influence 
of  the  blood  fluids  in  this  phenomenon  has  been  the  subject  of  much 
study  and  conflicting  results.  Metchnikoff  and  his  co-workers  were  of 
the  opinion  that  the  leucocytes  contain  complement,  which,  as  has  been 
shown  in  previous  chapters,  is  required  for  the  action  of  bactericidal 
and  bacteriolytic  amboceptors.  They  believed  that  this  complement  is 
liberated  only  upon  the  destruction  of  the  leucocytes  as  seen  in  phag- 
olysis  for  they  were  unable  to  find  complement  in  plasma.  They  inter- 
preted the  presence  of  complement  in  serum  as  due  to  the  death  of  the 
leucocytes  during  clotting  of  the  blood.  This  interpretation  has  been 
combated  by  numerous  observers  who  have  been  able  to  demonstrate 
complement  in  plasma.  In  support  of  the  conception  that  the  death  of 
the  bacteria  is  due  to  completion  of  the  bactericidal  amboceptor-antigen 
complex  by  complement  in  the  leucocyte,  is  Bordet's  work  with  cholera 
vibrios.  Using  immune  sera  which  contained  bacteriolytic  amboceptor, 
he  found  no  lysis  except  in  those  bacteria  that  were  within  phagocytic 
cells.  As  opposed  to  this  conception,  the  work  of  Neufeld  and  his 
collaborators  has  shown  that  sera  may  be  richly  opsonic  without  con- 
taining lytic  amboceptors,  and  in  these  instances  the  bacteria  are 
destroyed  and  digested  by  the  phagocytes.  The  destruction  varies  with 
different  organisms  and  with  the  virulence  of  the  organisms,  the  more 
virulent  being  less  readily  killed  than  the  avirulent  strains.  Bacteria 
may  be  cultivated  on  artificial  media  after  having  been  ingested,  a 
certain  amount  of  time  being  necessary  to  kill  the  organisms.  The 
act  of  digestion  is  closely  bound  up  with  that  of  killing  the  organisms. 
The  presence  of  a  proteolytic  ferment  in  leucocytes  has  been  known 


CELLULAR  RESISTANCE  167 

since  the  work  of  Mueller  and  Jochmann,  who  placed  the  leucocytes 
of  animals  upon  plates  similar  to  those  used  for  bacterial  cultivation. 
At  incubator  temperature,  the  leucocytes  exhibit  distinct  proteolytic 
power.  Recent  studies  by  Van  Calcar  would  appear  to  indicate  that 
the  organs  of  the  body  which  secrete  digestive  ferments  have  some 
influence  over  the  ferments  existing  within  the  leucocytes.  He  found, 
for  example,  that  after  the  removal  of  the  stomach  the  leucocytes  of 
the  animal  were  unable  to  act  as  peptic  digesters.  Similarly  the 
removal  of  the  pancreas  destroys  the  ability  of  the  leucocytes  to  act 
as  tryptic  digesters.  In  summary  it  is  necessary,  in  order  to  accom- 
plish destruction  and  digestion,  to  sensitize  the  organisms  and  to  have 
present  active  living  leucocytes.  Opsonization  will  not  in  itself  kill  or 
digest  the  organisms;  therefore,  the  phagocyte  must  furnish  some 
substance  which  either  completes  the  action  of  the  opsonin  or  of  itself 
can  kill  and  digest  the  organisms.  The  fact  that  phagocytosis  in  all 
its  stages  may  occur  in  slight  degree  independently  of  opsonin  would 
indicate  that  the  phagocyte  is  the  important  element  in  death  and  diges- 
tion of  the  phagocyted  object.  Recent  work  by  Bachmann  would  indicate 
that  the  leucocytes  of  normal  and  immune  animals  have  a  different 
capacity  for  protecting  against  disease.  Sixty  times  more  leucocytes 
from  a  normal  animal  were  needed  to  save  a  guinea-pig  against  typhoid 
infection  than  the  number  required  from  an  immune  animal.  In  the 
case  of  anthrax  the  leucocytes  from  immune  animals  were  eighty  times 
more  active  than  those  from  normal  animals.  That  these  studies  can 
be  interpreted  as  indicating  a  variation  in  the  actual  phagocytic  power 
of  leucocytes  is  open  to  considerable  question. 

It  is  probable  that  the  affinity  of  the  phagocyte  and  phagocytable 
object  is,  in  large  part  if  not  entirely,  a  physical  chemical  phenomenon 
entered  into  on  the  one  hand  by  the  cytoplasm  of  the  leucocyte  and 
other  cells  and,  on  the  other  hand,  the  opsonized  organisms  or  other 
object.  The  ingestion,  death  and  digestion  are  dependent  upon  the  life 
function  of  the  phagocyte,  which  is  capable  of  liberating  a  microbicidal 
and  microbilytic  substance  capable  of  combining  with  the  microorgan- 
ism to  bring  about  its  death  and  destruction. 

OTHER  MANIFESTATIONS  OF  CELLULAR  RESISTANCE 

Introduction. — rStudies  of  inflammation  and  of  other  cellular  activ- 
ities have  made  it  clear  that  body  cells  play  an  important  part  in  resist- 
ance to  disease  that  is  not  entirely  explained  by  the  phagocytic  capacity 
of  certain  of  the  cells.  As  has  been  indicated,  cells  other  than  the 
polymorphonuclear  leucocyte  and  the  large  mononuclear  cell  possess 
the  property  of  phagocytosis,  but  this  is  occasional  and  presumably 
not  of  great  importance.  It  seems  desirable,  however,  to  discuss  the 
mechanisms  of  resistance  as  influenced  by  properties  of  the  leucocytes 
other  than  phagocytosis,  by  activities  of  the  lymphocytes  and  by  the 
cells  and  fluids  which  play  a  part  in  inflammation. 

Bactericidal  Extracts  of  Leucocytes. — The  destruction  of  bacteria 
within  the  phagocyte  so  impressed  Metchnikoff  that  he  assumed  that 


1 68  THE  PRINCIPLES  OF  IMMUNOLOGY 

extracellular  destruction  is  accomplished  by  identical  destructive 
agents.  The  demonstration  that  extracellular  destruction  of  bacteria 
(bacteriolysis)  requires  the  participation  of  amboceptor  and  comple- 
ment had  little  influence  on  Metchnikoff's  views,  inasmuch  as  he  was 
convinced  that  complement  originates  solely  in  the  leucocytes.  As  we 
have  stated  (page  129)  the  more  recent  examination  of  this  problem 
makes  it  certain  that  complement  exists  free  in  the  blood.  Further 
study,  more  particularly  of  opsonins  and  bacteriotropins,  has  made 
it  apparent  that  the  mechanism  of  intracellular  digestion  is  quite  differ- 
ent from  that  of  extracellular  lysis.  Nevertheless,  the  leucocytes  may 
contribute  to  the  extracellular  destruction  of  bacteria.  Buchner  showed 
that  the  exudation,  produced  in  the  pleura  of  rabbits  and  dogs  by  injec- 
tions of  aleuronat,  removed  and  killed  by  freezing  and  thawing,  pos- 
sesses the  property  of  killing  bacillus  coli.  Denys  and  Kaisin  produced 
pleural  exudates  by  injection  of  killed  staphylococci  and  removed  the 
cells  by  centrifugation.  The  clear  supernatant  fluid  was  actively  bac- 
tericidal. Others  have  made  extracts  of  exudates,  and  of  leucocytes 
obtained  from  the  blood,  and  have  demonstrated  that  a  bactericidal 
substance  is  to  be  obtained.  Certainly  these  substances  are  yielded  up 
after  the  destruction  of  the  cells  and,  according  to  Petterson,  they 
may  be  secreted  by  the  cell  during  its  life.  The  substances  are  resistant 
to  a  temperature  of  56°  C,  but  after  inactivation  by  heat  to  75°  to  80° 
C.  they  cannot  be  reactivated  by  the  addition  of  fresh  extracts.  This 
substance  or  group  of  substances  has  been  called  endolysin  by  Petterson 
and  leucine  by  Schneider.  It  is  not  identical  in  all  animals  since  that 
from  dogs,  rabbits  and  guinea-pigs  kills  bacillus  proteus  and  bacillus 
anthracis,  but  that  from  the  guinea-pig  and  cat  fail  to  kill  the  bacillus 
typhosus  and  the  spirillum  cholerae. 

Bachmann  has  recently  reported  on  a  so-called  leucocyte  antibody, 
"  cmticorps  leucocytaire"  which  is  distinct  from  the  bactericidal  endo- 
lysin. It  appears  in  the  leucocytes  of  immunized  animals  and  may  serve 
to  produce  passive  immunity  in  other  animals.  It  is  found  only  in  the 
polymorphonuclear  leucocytes  and  may  be  extracted  in  normal  serum. 
It  is  effective  in  protecting  guinea-pigs  against  intraperitoneal  injection 
of  the  specific  organism  and  also  acts  beneficially  and  specifically  upon 
established  infections.  A  temperature  of  75°  C.  destroys  this  substance, 
but  if  the  material  is  well  diluted  and  gelatin  added,  the  same  degree 
of  heat  serves  to  destroy  the  non-specific  bactericidal  substances  (endo- 
lysins)  but  permits  the  specific  leucocyte  antibody  to  remain  active. 
Bachmann  believes  that  the  persistence  of  this  antibody  in  the  leuco- 
cytes explains  the  fact  that  individuals  retain  immunity  to  certain 
diseases  after  the  serum  antibodies  are  no  longer  demonstrable. 

Leucocyte  Enzymes. — In  contrast  to  the  bactericidal  substances 
extracted  from  leucocytes  it  is  possible  to  obtain  enzymes.  Leber,  in 
a  study  of  inflammation,  found  that  sterile  pus  can  liquefy  gelatin  and 
the  study  of  this  proteolytic  enzyme,  the  leucoprotease,  has  been  ex- 
tended by  Miiller  and  Jochmann,  Opie,  Longcope  and  others.  This 
leucoprotease  may  be  purified  by  precipitation  with  alcohol  more  par- 


CELLULAR  RESISTANCE  169 

ticularly  from  glycerol  extracts  and  the  desiccated  precipitate  may  be 
preserved  almost  indefinitely.  In  the  moist  state  temperatures  of  from 
50°  to  65°  C.  increase  its  activity,  but  at  70°  to  75°  C.  it  is  destroyed. 
It  acts  best  in  weakly  alkaline  or  neutral  medium,  and  is  inhibited  by 
acid.  It  differs  from  trypsin  in  that  it  is  much  less  active ;  it  does  not 
require  activation  by  any  such  substance  as  enterokinase,  and  exists 
within  the  cells  in  an  active  state  rather  than  in  the  form  of  zymogen. 
It  differs  from  the  bactericidal  extracts  in  that  it  cannot  kill  bacteria, 
but  may  digest  them  after  their  death.  The  blood  possesses  an  anti- 
enzyme,  but  when  the  cells  accumulate  in  bulk,  as  in  the  case  of  inflam- 
matory exudates,  the  anti-enzyme  is  overbalanced  and  the  protease 
dissolves  necrotic  cells,  dead  bacteria  and  other  detritus.  It  is  of 
considerable  importance  in  the  resolution  of  lobar  pneumonia.  In 
addition  the  leucocytes  are  stated  to  contain  amylase,  diastase,  catalase, 
oxidase,  peroxidase,  nuclease  and  an  ereptic  ferment,  but  there  appears 
to  be  a  difference  of  opinion  in  regard  to  lipase. 

Opie  has  described  an  additional  ferment  in  areas  rich  in  large 
mononuclear  cells,  which  acts  best  in  a  very  weak  acid  medium.  It  is 
inhibited  by  temperatures  of  50°  to  65°  C.,  by  alkali  and  by  the  con- 
centration of  HC1  (0.2  per  cent.)  favorable  for  the  action  of  pepsin. 
He  was  able  to  demonstrate  this  ferment  in  hyperplastic  lymph-nodes 
rich  in  large  mononuclear  phagocytes.  It  is  closely  related  to  the 
enzymes  of  tissue  autolysis.  The  acid  medium  which  favors  the  action 
of  this  enzyme  inhibits  the  activity  of  anti-enzyme. 

Leucocyte  Extracts  for  Therapeutic  Purposes. — Petterson  noted 
that  when  leucocytes  are  placed  in  contact  with  blood  serum  for  several 
hours  the  mixture  is  more  actively  bactericidal  than  the  serum  alone 
or  salt  solution  extracts  of  the  leucocytes.  This  led  to  experiments  in 
which  he  injected  leucocytes  simultaneously  with  anthrax  bacilli  into 
dogs  and  found  a  moderate  protection  by  this  treatment.  Opie  similarly 
observed  that  the  injection  of  leucocytes  and  tubercle  bacilli  into  the 
pleura  of  dogs  led  to  less  severe  manifestations  than  when  tubercle 
bacilli  alone  are  injected.  Probably  the  most  important  contributions 
to  the  treatment  of  disease  by  leucocyte  extracts  are  the  studies  of  Hiss 
with  the  collaboration  of  Zinsser,  Dwyer  and  others.  Hiss  obtained  the 
leucocytes  from  pleural  exudates  produced  by  the  injection  of  aleuronat 
suspensions.  This  was  centrifuged  before  clotting  occurred  and  the 
cells  emulsified  in  distilled  water.  Either  the  leucocytes  or  the  leu- 
cocytes and  supernatant  fluid  were  employed  for  treatment.  From 
experiments  with  staphylococcus,  pneumococcus,  streptococcus,  meningo- 
coccus  and  typhoid  bacillus  infections  in  rabbits,  it  was  determined  that 
protection  was  afforded  by  the  extracts  and  that  the  infection  was 
favorably  influenced  if  therapeutic  doses  were  given  as  late  as  twenty- 
four  hours  after  infection.  Encouraging  results  were  also  obtained  in 
the  treatment  of  human  cases  of  pneumonia,  meningitis,  staphylococcus 
infections,  erysipelas  and  other  diseases.  In  analyzing  the  beneficial 
effects  of  this  form  of  treatment,  it  was  found  that  the  bactericidal 
properties  of  the  extracts  are  not  sufficiently  great  to  explain  their 


170  THE  PRINCIPLES  OF  IMMUNOLOGY 

influence,  they  do  not  materially  favor  phagocytosis  but  appear  to 
augment  the  migration  of  leucocytes  to  a  slight  degree  and  possibly  are 
of  importance  in  this  way  because  of  the  fact  that  they  exert  positive 
chemotaxis.  Zinsser  states  "  we  are  inclined  to  believe  at  present  that 
the  beneficial  effects  of  leucocyte  extracts  are  based  on  the  same  prin- 
ciples as  those  which  determine  the  reactions  following  on  the  injection 
of  bacterial  and  any  other  protein."  To  us  it  appears  that  this  method 
is  to  be  included  in  the  category  of  non-specific  therapy  previously 
discussed  (page  30). 

Specific  Hyperleucocytosis. — Following  upon  the  earlier  sug- 
gestion of  Bordet,  Gay  and  his  collaborators  found  that  immune  animals 
exhibit  a  much  higher  degree  of  leucocytosis  following  the  injection 
of  the  organism  to  which  they  had  been  immunized  than  do  normal 
animals.  For  example,  rabbits  immunized  to  typhoid  bacilli  reacted 
to  subsequent  injections  of  typhoid  bacilli  with  blood  counts  of  as  high  as 
150,000  leucocytes  per  cmm.,  whereas  normal  rabbits  showed  a  reaction 
of  only  40,000  to  50,000  leucocytes  per  cmm.  This  phenomenon  of  spe- 
cific hyperleucocytosis  has  been  contradicted  by  McWilliams,  who  found 
no  important  difference  in  response  between  normal  and  immune  animals 
and  further  states  that  typhoid  immune  rabbits  react  in  essentially  the 
same  degree  to  colon  bacilli  as  to  typhoid  bacilli.  Others  have  confirmed 
the  work  of  McWilliams.  Zinsser  and  Tsen  found  a  slight  favorable 
difference  in  animals  immunized  to  Gram  negative  cocci  and  a  somewhat 
more  marked  difference  in  those  immunized  to  Gram  positive  cocci,  not 
in  any  case,  however,  to  the  degree  indicated  by  Gay.  There  seems 
little  reason  for  believing  that  a  specific  hyperleucocytosis  plays  any 
important  part  in  resistance  to  infection.  This,  however,  is  not  to  be 
construed  as  an  argument  against  vaccination,  since  the  latter  procedure 
is  important  in  the  production  of  specific  opsonins,  agglutinins  and  other 
immune  bodies.  Any  response  to  vaccination  in  the  form  of  leucocytosis 
must  be  regarded  as  only  in  small  part  if  at  all  specific  and  is  probably 
of  the  same  nature  as  the  leucocytic  response  to  the  injection  of  non- 
specific proteins  and  their  products. 

The  Lymphocytes. — Lymphocytes  appear  in  inflammatory  areas  as 
the  result  of  infection,  but  accumulate  in  largest  amounts  in  chronic 
inflammatory  areas  where,  in  most  instances,  the  active  infective  agent 
is  no  longer  present.  The  part  they  play  in  the  phenomenon  of  inflam- 
mation and  in  protection  against  infection  is  not  understood.  From 
the  work  of  Opie  it  seemsi  probable  that  the  lymphocytes  may  be,  in 
part,  the  source  of  the  ferment  which  he  describes  as  operating  in 
weakly  acid  media.  As  pointed  out  above,  this  ferment  was  obtained 
from  hyperplastic  lymph-nodes.  The  lymphocytes  are  said  to  contain 
a  lipase,  and  it  is  suggested  that  the  large  collections  of  these  cells 
about  tuberculous  foci  may  serve  by  the  action  of  the  lipase  to  break 
down  the  waxy  shell  of  the  bacilli.  The  lymphocyte  is  stated  to  possess 
phagocytic  properties,  but  these  are  at  best  very  slight  and  probably 
play  no  important  part  in  resistance  to  disease.  It  has  long  been  noted 
that  the  presence  of  tumors  in  the  body  often  excites  a  neighboring 


CELLULAR  RESISTANCE  171 

chronic  inflammatory  reaction  in  which  lymphocytes  appear  in  con- 
siderable numbers.  J.  B.  Murphy  and  his  collaborators  have  put  to 
the  test  of  experiment  the  hypothesis  that  lymphocytes  are  of  import- 
ance in  resistance  to  cancer.  By  the  use  of  the  X-ray  they  were  able 
to  destroy  practically  all  the  lymphoid  tissue  of  the  body  of  animals  and 
found  in  these  animals  a  decreased  resistance  to  transplanted  cancer. 
Immunity  already  established  to  cancer  was  also  destroyed  by  this 
procedure.  Similarly  there  was  a  lowered  resistance  to  tuberculosis 
and  to  anterior  poliomyelitis.  In  tuberculosis  the  lymphocyte  constitutes 
a  large  element  in  the  inflammatory  reaction,  and  this  is  true  also  in  the 
later  stages  of  acute  anterior  poliomyelitis.  Although  small  doses  of 
X-ray  may  stimulate  lymphocyte  production,  Murphy  and  his  asso- 
ciates found  that  dry  heat  produces  a  more  durable  increase  in  the 
circulating  lymphocytes.  By  increasing  the  lymphocytes  in  this  fashion 
they  demonstrated  "  the  establishment  of  a  high  degree  of  immunity 
to  certain  transplantable  cancers  in  mice,"  regardless  of  whether  these 
cancers  naturally  showed  a  high  or  low  percentage  of  successful  inocula- 
tion. The  same  was  found  to  be  true  in  regard  to  the  implantation  of 
grafts  from  spontaneous  cancers  into  the  animals  from  which  the 
grafts  were  removed.  This  subject  has  also  been  studied  by  F.  C. 
Wood  and  associates  in  the  Crocker  Laboratory.  They  found  that  mice 
with  lymphatic  leucemia  show  no  demonstrable  immunity  to  tumors. 
They  found  that  reduction  of  the  total  leucocyte  count  by  means  of 
X-ray  or  radium  produces  no  increase  in  the  successful  transplanta- 
tion of  normal  tissues.  They  found  further  that  successful  transplan- 
tation of  the  guinea-pig  fibrosarcoma  is  not  influenced  by  the  use  of 
X-ray.  They  selected  an  immune  strain  of  rats,  exposed  them  to  X-ray 
and  found  no  change  in  susceptibility  to  transplantable  tumors.  They 
found  that  the  use  of  X-ray  on  rats  in  which  a  highly  virulent  tumor  had 
been  implanted  did  not  prolong  the  life  of  the  tumor.  Wood  states 
that  "  it  is,  therefore,  evident  that  the  lymphocyte  is  in  no  way  corre- 
lated with  cancer  immunity."  Sittenfield  also  found  that  artificial 
lymphocytosis  has  no  effect  whatever  on  tumor  growth.  It  is  of  further 
interest  that  in  human  cancer  the  lymphocytes  collect  about  the  slowly- 
growing  rather  than  the  rapidly-growing  tumors  and  that  the  metastases 
are  frequent  in  the  lymph-nodes.  The  later  experiments  of  Murphy 
on  the  lymphocytosis  induced  by  heat  have  not  received  as  yet  extensive 
examination ;  therefore,  the  question  remains  open.  Murphy's  experi- 
ments are  so  well  conducted  that  it  is  difficult  to  be  assured  that  the 
lymphocytes  play  no  part.  The  work  of  Wood  carried  out  on  a  large 
number  of  animals  is  of  especial  significance  and  would  indicate  that 
the  lymphocyte  plays  no  such  important  part  in  resistance  to  cancer  as 
Murphy's  work  appears  to  indicate. 

Platelets. — In  1901  Levaditi  noticed  that  following  the  injection  of 
cholera  vibrios  they  were  often  found  clumped  around  small  masses 
of  platelets.  The  phenomenon  was  called  thigmotropism.  Govaerts 
subsequently  demonstrated  that  the  clumping  is  influenced  by  the  action 
of  opsonins.  LeFevre  found  that  anti-bacterial  immunization  increases 


172  THE  PRINCIPLES  OF  IMMUNOLOGY 

thigmotropism  because  of  the  increase  in  activity  of  opsonins.  Further 
study  may  throw  light  on  the  mechanism  of  the  process,  but  at  present 
its  function  is  obscure. 

The  Influence  of  Inflammation. — Infection  always  produces  some 
degree  of  inflammatory  reaction,  but  this  varies  considerably  with  the 
type  of  infectious  organism  and  with  the  capacity  of  the  host  to  react. 
The  exudate  comprises  the  polymorphonuclear  leucocyte,  the  lympho- 
cyte, the  plasma  cell,  the  large  mononuclear  cell,  certain  other  less 
important  cells,  the  red  blood-corpuscles,  serum  and  fibrin.  The  part 
played  by  the  more  important  of  these  cells  is  indicated  above.  As  far 
as  we  can  determine,  the  red  blood-corpuscles  appear  more  as  an 
accident  of  the  process  than  as  an  essential  part  of  it.  The  fluid  part  of 
the  exudate  rapidly  coagulates  with  the  formation  of  fibrin  and  serum. 
There  can  be  no  doubt  that  the  serum  serves  in  certain  measure  to 
concentrate  in  the  inflammatory  areas  those  immune  bodies  qualified 
to  offer  resistance  to  the  invader  and  its  products.  In  case  toxic 
products  are  present,  these  are  diluted  by  the  serum  and  the  subsequent 
absorption  of  the  serum  with  this  diluted  poison  aids  in  its  elimination 
from  the  body.  The  fibrin  network  probably  serves  in  a  certain  measure 
to  wall  off  and  limit  the  growth  of  the  invading  organism.  It  also 
serves  as  a  scaffolding  for  the  support  of  newly-growing  fixed  tissue. 
Very  early  in  the  course  of  an  acute  inflammation  the  connective  tissue 
cells  proliferate.  They  may  be  phagocytic,  but  this  property  is  of 
little  significance.  Certainly  the  most  important  function  of  the  con- 
nective tissue  in  resistance  to  infection  is  the  formation  of  a  tissue 
which  serves  to  limit  the  advance  of  the  infection.  The  newly-growing 
connective  tissue,  with  its  capillaries,  constitutes  granulation  tissue  and 
the  resistance  of  granulation  tissue  to  infection  is  a  matter  of  common 
observance.  As  the  inflammation  becomes  chronic  the  connective  itssue 
becomes  denser  and  thereby  provides  a  much  less  permeable  wall  than 
is  found  in  the  earlier  stages  of  the  process.  The  production  of  a  local 
inflammation  leads  to  the  formation  of  an  exudate  which  by  virtue  of 
the  polymorphonuclear  leucocytes  opposes  to  infection  the  important 
process  of  phagocytosis;  the  liberation  of  bactericidal  substances  and 
of  enzymes  from  the  leucocytes  serves  to  aid  in  resistance  and  to  liquefy 
dead  tissues  and  dead  bacteria.  Under  favorable  circumstances  addi- 
tional enzymes  are  provided  by  the  large  monuclear  cells  and  lympho- 
cyte. The  large  mononuclears  aid  in  the  removal  of  dead  material  by 
virtue  of  their  phagocytic  powers.  The  fluid  part  of  the  exudate  brings 
into  the  process  the  immune  bodies  of  the  circulating  blood,  serves  to 
dilute  toxic  products  and  favors  their  absorption  and  elimination  in 
dilute  form.  The  fibrin,  granulation  tissue  and  cicatrization  act  as  de- 
limiting elements  and  operate  toward  the  localization  of  the  process. 


CHAPTER  VIII 
COMPLEMENT  FIXATION 

INTRODUCTION. 

THE  BORDET-GENGOU  PHENOMENON. 
LABORATORY  DEMONSTRATION. 

ANTI-COMPLEMENTARY  AND  HEMOLYTIC  TITER  OF  ANTIGEN. 
THE  TEST. 

SPECIFIC  CHARACTER  OF  THE  TEST. 
INHIBITION  ZONES. 
GROUP  REACTIONS. 

RELATION  OF  COMPLEMENT-FIXING  BODIES  TO  OTHER  IMMUNE  BODIES. 
IS  THE  COMPLEMENT-FIXING  BODY  AN   AMBOCEPTOR  ? 
ACTIVATION   BY    COMPLEMENT. 

FIXATION  OF  THE  COMPLEMENT  OF  NATURAL  HEMOLYSINS. 
INHIBITION   OF   COMPLEMENT  OTHER  THAN   BY   FIXATION. 
ANTI-COMPLEMENTARY  CHEMICAL  AGENCIES. 

ANTI-COMPLEMENTARY  ACTION   OF  CELLS,   TISSUE  EXTRACTS   AND  BODY  FLUIDS. 
ANTI-COMPLEMENTARY   ACTIVITY   OF  IMMUNE    SERA. 

Introduction. — A  summary  of  the  hypotheses  concerning  the  con- 
stitution of  complements  shows  that  there  are  three  important  views 
offered,  namely  the  "  pluralistic  "  conception  of  Ehrlich  and  Morgen- 
roth,  the  "  dualistic "  of  Metchnikoff  and  "  unitaristic "  of  Bordet. 
As  has  been  explained,  the  view  of  Metchnikoff  that  complement  might 
be  a  "  macrocytase  "  or  a  "  microcytase  "  depending  upon  its  cellular 
origin  has  been  abandoned  by  most  immunologists.  Thus  the  conflict 
has  been,  and  in  certain  measure  still  is,  between  the  views  of  Ehrlich 
and  of  Bordet.  Bordet  and  Gengou  in  demonstrating  that  the  same 
complement  is  called  on  for  bacteriolysis  as  for  hemolysis,  discovered 
the  phenomenon  named  by  them  complement  fixation  ("  la  fixation 
d'alexine  ")  which  we  employ  in  sharp  contradistinction  to  complement 
deviation.  The  latter  term  implies  the  anchoring  of  complement  by 
free  amboceptor  units,  whereas  fixation  signifies  the  entrance  of  the 
complement  into  combination  with  antigen  and  amboceptor.  In  brief, 
they  showed  that  if  complement  is  utilized  in  the  process  of  bacteriolysis 
it  is  not  available  for  hemolysis. 

The  Bordet-Gengou  Phenomenon.-^The  primary  experiment  was 
performed  with  plague  bacilli,  the  serum  of  a  horse  immunized  to 
plague  bacilli,  fresh  guinea-pig  serum  (complement)  and  sensitized  red 
blood-corpuscles,  i.e.,  corpuscles  saturated  with  a  specific  hemolytic 
immune  serum.  They  mixed  an  emulsion  of  plague  bacilli,  the  anti- 
plague  horse  serum  and  complement.  This  mixture  was  left  at  room 
temperature  for  five  hours  and  then  the  previously-sensitized  erythro- 
cytes  added,  the  mixture  incubated  and  observed.  No  hemolysis 
appeared,  although  the  corpuscles  were  often  agglutinated  by  the  hemo- 
lytic (and  hemagglutinative)  immune  serum.  Naturally,  such  an  ex- 

173 


174  THE  PRINCIPLES  OF  IMMUNOLOGY 

periment  required  numerous  controls,  the  complete  series  being  indi- 
cated in  the  following  protocol : 

1.  Plague    bacilli    -j-    immune  horse  serum  -f-  complement  +v  sensitized  cells  = 

No  hemolysis. 

2.  Plague    bacilli    +     normal  horse  serum  -f  complement  +  sensitized  cells  = 

Hemolysis. 

3.  —    —    —    —      immune  horse  serum  -f~  complement  +  sensitized  cells  — 
Hemolysis. 

4.  —    —    —    —       normal  horse  serum  +  complement  +  sensitized  cells  = 
Hemolysis. 

5.  Plague    bacilli    -f-    immune  horse  serum —  —     -f*  sensitized  cells  = 

No  hemolysis. 

6.  Plague    bacilli    +      normal  horse  serum +  sensitized  cells  = 

No  hemolysis. 

Throughout  the  experiment  all  the  sera  were  inactivated  except  the 
fresh  guinea-pig  complement  and  all  mixtures  stood  at  room  tempera- 
ture for  five  hours  before  the  addition  of  the  sensitized  erythrocytes. 
Hemolysis  in  tube  2  shows  that  normal  horse  serum  does  not  serve 
as  an  amboceptor  or  sensitizer  for  the  plague  bacilli  and  therefore 
does  not  prevent  the  complement  from  entering  into  combination  with 
the  sensitized  erythrocytes.  Tubes  3  and  4  contain  no  bacterial 
antigen,  cannot  utilize  complement  and  therefore  hemolysis  appears. 
Tubes  5  and  6  show  that  the  bacteria  are  not  hemolytic  and  that 
neither  of  the  inactivated  immune  sera  nor  the  inactivated  normal  horse 
serum  contain  complement  for  the  completion  of  the  amboceptor-cell 
complex.  Bordet  and  Gengou  showed  that  the  same  phenomenon  could 
be  observed  with  a  wide  variety  of  bacteria  and  specific  immune  sera 
both  of  human  and  lower  animal  origin;  these  operate  to  fix  both 
guinea-pig  and  human  complements,  so  as  to  prevent  combination  of 
these  complements  with  hemolytic  immune  sera  from  several  species. 
Furthermore,  the  immune  sera  so  fixed  might  be  specific  for  several 
varieties  of  erythrocytes.  Muir  and  Martin  found,  however,  that 
whereas  most  complements  can  be  fixed  in  such  experiments,  this  is  not 
universally  true  and  occasional  complements  are  met  with  which  do 
not  enter  into  certain  combinations.  Furthermore,  the  process  could 
•be  reversed  so  that  the  fixation  of  complement  in  hemolysis  prevented 
its  action  to  bring  about  bacteriolysis  of  sensitized  bacteria.  Thus  it 
appeared  that  one  and  the  same  complement  operates  for  the  produc- 
tion of  both  bacteriolysis  and  hemolysis.  This  demonstration  of  the 
unity  of  complement  has  been  combated  by  later  work,  and  it  now 
appears  that  there  are  certain  exceptions  to  the  rule,  although  it  can 
generally  be  accepted. 

Laboratory  Demonstration  of  the  Bordet-Gengou  Phenomenon. — In 
order  to  demonstrate  the  phenomenon  it  is  not  necessary  to  use  plague  bacilli,  as 
others  serve  the  purpose  equally  well.  The  readily  obtainable  typhoid  bacillus 
and  typhoid  immune  serum  can  be  used  with  good  results.  In  setting  up  the  test 
it  is  important  to  bear  in  mind  the  fact  that  numerous  substances  may  interfere 
with  the  activity  of  complement,  and  among  these  are  certain  concentrations  of 


COMPLEMENT  FIXATION 


175 


bacterial  emulsions  and  extracts.  Therefore,  it  is  necessary  to  be  sure  that  the 
amount  of  bacterial  emulsion  used  in  the  test  is  not  "  anti-complementary,"  but 
yet  in  sufficient  concentration  to  operate  well.  The  emulsion  is  made  from  a 
twenty-four-hour  slant  agar  culture  (see  page  81  for  preparation)  and  may  be 
killed  by  heat  or  formalin.  The  preliminary  titration  may  be  set  up  as  follows : 


Bacterial 
emulsion 

Complement 
i-io  dilution 

1 

Hemolytic 
amboceptor 

Erythrocyte 
suspension 

I 

Result 

0.5  c.c. 
0.4  c.c. 
0.3  c.c. 

0.2   C.C. 
O.I    C.C. 

o  5  c.c 

0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 

incubate  one 

0.5  c.c.  (2  doses) 
0.5  c.c.  (2  doses) 
0.5  c.c.  (2  doses) 
0.5  c.c.  (2  doses) 
0.5  c.c.  (2  doses) 
0.5  c.c.  (2  doses) 

0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 

incubate  one 

P.H. 
C.H. 
C.H. 
C.H. 

Each  tube  should  be  made  up  to  a  volume  of  2.0  c.c.  with  salt  solution  before 
primary  incubation.  If  convenient  the  erythrocytes  may  be  sensitized  by  the 
previous  addition  of  amboceptor.  In  the  protocol  C.H.  indicates  complete 
hemolysis,  P.H.  partial  hemolysis  and  —  no  hemolysis. 

The  Test. — The  results  given  indicate  that  0.5  c.c.  bacterial  emulsion  is  defi- 
nitely anti-complementary,  but  the  0.3  c.c.  has  no  such  influence.  The  last  tube 
excludes  hemolytic  activity  on  the  part  of  the  emulsion.  In  order  to  be  abso- 
lutely sure  that  the  final  test  will  not  be  misleading  through  the  anti-comple- 
mentary action  of  the  bacterial  emulsion  it  is  advisable  to  use  the  next  smaller 
amount  than  the  titration  shows  to  be  free  of  anti-complementary  activity,  which 
in  this  case  is  0.2  c.c.  This  being  the  case  2.0  c.c.  bacterial  emulsion  may  be 
diluted  with  3.0  c.c.  salt  solution,  whereupon  0.5  c.c.  of  the  dilution  will  contain 
0.2  c.c.  original  emulsion.  The  complement-fixation  test  may  then  be  set  up 
as  follows : 


Bacterial 
emulsion 
(2-5) 

Anti- 
typhoid 
immune 
serum 

Normal 
rabbit 
serum 

Complement 

Salt 
solution 

L 

Sensitized 
erythrocytes 

£ 

Results 

O  5  C.C. 

(l-IO 

dilution) 

O.5  C.C. 

(l-IO 

dilution) 

(l-IO 

dilution) 
0.5  c.c. 

1 

1 

I.O  C.C. 

& 

a> 

0.5  c.c. 
0.5  c.c. 

0.5  c.c. 

0.5  c.c. 
0.5  c.c. 

0.5  c.c. 

<D 

I.O  C.C. 
I.O  C.C. 

$ 
1 

C.H. 
C.H. 

0.5  c.c 

0.5  c.c. 

0.5  c.c. 

I 

I.O  C.C. 

C.H. 

0.5  c.c. 

0.5  c.c. 

0.5  c.c. 

£ 

•  I.O  C.C. 

a 

C.H. 

0.5  c.c. 

I.O  C.C. 

I.O  C.C. 

C.H. 

1.5  c.c. 

I  .O  C.C. 

The  first  incubation  permits  of  fixation  of  the  complement  by  the  bacteria 
and  their  specific  immune  serum  and  the  second  determines  whether  or  not 
complement  is  free  to  act  upon  the  sensitized  erythrocytes.  For  sensitization  of 
erythrocytes  the  hemolytic  immune  serum  should  be  diluted  so  that  0.5  c.c. 
contains  two  units  hemolytic  amboceptor,  then  added  to  an  equal  volume  5  per 
cent,  erythrocyte  suspension.  In  the  above  test  the  immune  serum  is  diluted,  so 
that  2.5  c.c.  contain  ten  units  amboceptor;  it  is  then  added  to  2.5  c.c.  5  per  cent, 
erythrocyte  suspension  and  the  mixture  allowed  to  remain  at  room  temperature 
for  one  hour.  The  protocol  given  above  shows,  reading  from  below  upward, 
that  the  hemolytic  immune  serum  used  for  sensitization  is  not  of  itself  hemolytic, 
that  the  complement  is  in  sufficient  concentration  for  hemolysis,  that  neither  the 
bacterial  emulsion  nor  the  typhoid  immune  serum  is  anti-complementary  in  the 
amounts  used.  In  the  first  tube  the  bacterial  emulsion,  specific  anti-bacterial 
serum  and  complement  interact  so  that  the  complement  is  not  free  to  combine  with 
the  sensitized  erythrocytes,  whereas  tube  2  shows  that  normal  rabbit  serum  will 
not  fix  complement. 

Specific  Character  of  the  Test. — In  order  to  elaborate  the  test  and  to  show 
its  specificity  it  is  well  also  to  titrate  an  emulsion  of  some  other  organism,  for 
example,  colon  bacilli  for  anti-complementary  activity  at  the  same  time  the 
typhoid  emulsion  is  titrated  and  in  the  same  manner.  If  this  shows  anti-comple- 


176 


THE  PRINCIPLES  OF  IMMUNOLOGY 


mentary  activity  in  a  dose  of  0.3  c.c.,  then  O.I  c.c.  is  used  in  the  te<st.    The  fully 
controlled  test  would  then  be  set  up  as  follows  : 


Typhoid 

emulsion 

(2/5) 

Coli 
emulsion 
(i/5) 

Anti-typhoid 
immune  serum 
(i/io  dilution) 

Complement 
(i/io  dilu- 
tion) 

Salt  solu- 
tion 

u 

Sensitized 
erythro- 
cytes 

w, 

I 

Results 

0.5    c.c. 

0.5  c.c. 

0.5  c.c. 

rCJ 

.0  c.c. 

^H 

0.25  c.c. 

o.s  c.c. 

0.5  c.c. 
0.5  c.c. 

0.5  c.c. 
0.5  c.c. 

0.25  c.c. 

8 

o 

.0  c.c. 
.0  c  c 

CH 

0.5    c.c. 

0.5  c.c. 

0.5    c.c. 

ctf 

.0  c.c. 

3 

C.H. 

0.5  c.c. 

0.5  c.c. 

0.5    c.c. 

1 

.0  c.c. 

.g 

C.H. 

0.5  c.c. 

0.5  c.c. 

0.5    c.c. 

§ 

.0  c.c. 

i 

C.H. 

0.5  c.c. 

I.O     C.C. 

H-  1 

I.O  C  C. 

d 

CH 

I  e     c.C 

I  O  C  C 

•Reading  from  above  downward,  the  second  tube  shows  0.25  c.c.  typhoid 
emulsion  diluted  2  to  5,  thus  corresponding  in  bulk  of  original  emulsion,  to  the 
bulk  of  coli  'emulsion  in  0.5  c.c.  of  a  1-5  dilution.  It  is  necessary  to  use  the 
smaller  bulk  of  coli  emulsion  in  order  to  prevent  anti-complementary  activity. 
Both  quantities  of  typhoid  emulsion  are  sufficient  to  fix  the  complement,  whereas 
the  coli  emulsion  (tube  3)  does  not.  The  next  three  tubes  which  are  controls  show 
that  neither  typhoid  emulsion,  coli  emulsion  nor  anti-typhoid  immune  serum 
have  any  anti-complementary  activity.  The  last  two  tubes  show  that  the  com- 
plement is  in  sufficient  concentration  to  operate  and  that  the  sensitized  erythrc- 
cytes  will  not  of  themselves  hemolyze  under  the  conditions  of  the  experiment. 
If  the  results  prove  to  be  confusing  it  is  necessary  to  make  additional  controls 
to  determine  if  any  of  the  reagents  is  hemolytic.  This  contingency  is  extremely 
rare  if  proper  care  is  given  in  their  preparation.  The  test  in  this  form  shows 
that  the  reaction  is  specific. 

Inhibition  Zones. — The  phenomenon  of  complement  fixation  ex- 
hibits certain  of  the  characters  noted  in  regard  to  other  immune  reac- 
tions, not  only  in  the  titration  of  the  reacting  bodies  but  also  in  the 
formation  of  the  so-called  inhibition  zones  and  in  the  group  reaction. 
These  latter  features  are  best  illustrated  with  the  fixation  of  comple- 
ment by  immune  sera  prepared  from  the  use  of  dissolved  protein. 
Gengou,  a  year  after  the  publication  of  Bordet  and  Gengou,  showed 
that  the  inoculation  into  an  animal  of  dissolved  proteins,  such  as  egg- 
white,  could  lead  to  the  formation  of  bodies  which  participate  in  com- 
plement fixation  with  the  specific  antigen.  This  was  confirmed  by 
Moreschi  and  later  by  Neisser  and  Sachs.  The  latter  authors  applied 
the  reaction  to  the  forensic  determination  of  protein.  Gengou  was  of 
the  opinion  that  the  immunization  of  animals  with  dissolved  protein 
led  to  the  formation  not  only  of  precipitins  but  also  of  complement-fixing 
bodies.  The  relation  between  these  two  immune  substances  will  be 
discussed  after  presenting  data  concerning  inhibition  zones  and  group 
reaction.  An  experiment  from  the  work  of  Neisser  and  Sachs  serves 
to  illustrate  the  fact  that  immune  serum  may  be  used  in  the  reaction 
in  such  strong  concentration  as  to  inhibit  fixation  of  the  complement. 
For  this  purpose  they  arranged  two  series  of  tubes.  In  series  A  they 
placed  decreasing  amounts  of  the  specific  immune  serum,  a  constant 
quantity  of  0.2  c.c.  of  1-2000  solution  of  the  antigenic  human  serum 
and  o.i  c.c.  fresh  guinea-pig  complement.  In  series  B  the  same  con- 
stituents were  placed  with  the  exception  of  the  immune  serum  which 
was  replaced  in  each  tube  by  0.2  c.c.  salt  solution.  These  mixtures 
were  incubated  at  37°  C.  and  then  sensitized  red  blood-corpuscles  were 


COMPLEMENT  FIXATION  177 

added,  followed  by  another  period  of  incubation.  In  this  particular 
instance  they  employed  ox  blood-cells  and  immune  serum  prepared  by 
injection  of  ox  blood-cells  into  the  rabbit. 

MODIFIED  PROTOCOL  FROM  NEISSER  AND  SACHS 

Immune  serum  Complement  fixation 

i- 10  dilution  Series  A  Series  B 

I.O      C.C.  + 

0.75  c.c. 

0.5    c.c. 

0.35  c.c. 

0.25  c.c.  +++  — 

0.15  c.c.  ++ 

O.I      C.C.  +  — 

0.0      C.C.  — 

The  above  protocol  shows  that  in  the  strong  concentration  of  im- 
mune serum  the  fixation  of  complement  is  not  as  marked  as  in  some- 
what weaker  concentration.  Nevertheless,  there  also  comes  a  point 
when  the  concentration  is  too  dilute  to  permit  of  fixation.  The  tubes 
in  series  B  show  that  the  concentration  of  human  serum  in  itself  is  not 
sufficiently  great  to  prevent  the  hemolytic  reaction.  Two  points  are 
of  interest  in  this  connection.  In  the  first  place,  it  is  possible  to  add 
immune  serum  or  other  serum  in  amounts  so  large  that  the  serum  itself 
will  have  inhibitory  action  upon  the  complement.  Under  optimal  con- 
ditions immune  serum  may  be  diluted  to  an  extreme  degree  and  still  _ 
act  as  a  complement-fixing  body;  for  example,  Friedberger,  by  the 
use  of  a  well-prepared  serum  was  able  to  demonstrate  complement  ; 
fixation  by  an  immune  serum  diluted  1-1,000,000,000.  The  same  deli- 
cacy has  not  been  confirmed  by  other  investigators  and  must  be  regarded 
as  a  scientific  curiosity.  The  dilution  of  the  antigenic  protein  can  be 
carried  to  a  considerable  degree  but  not  usually  to  the  same  degree  as 
is  possible  with  antiserum. 

Group  Reactions. — In  the  application  of  the  complement-fixation 
test  to  the  forensic  determination  of  dissolved  protein,  Neisser  and 
Sachs  showed  that  the  group  phenomenon  also  appears.  They  also 
showed  that  the  antigenic  serum  could  be  very  much  reduced  in  amount 
and  still  give  complement  fixation.  The  following  protocol  illustrates 
the  manner  in  which  such  a  demonstration  may  be  made.  In  setting 
up  the  test  there  was  used  throughout  a  constant  quantity  of  o.i  c.c. 
immune  serum  prepared  by  the  injection  of  human  serum.  The  anti- 
genic serum  was  added  according  to  the  amounts  indicated  in  the 
protocol.  Complement  was  used  in  amounts  of  0.05  c.c.  The  mixtures 
were  incubated  and  then  beef  blood-corpuscles  which  had  been  sensitized 
with  a  specific  anti-beef  corpuscle  serum  were  added,  the  mixtures 
again  incubated  and  the  degree  of  fixation  determined. 

GROUP  REACTION  MODIFIED  FROM  NEISSER  AND  SACHS 

Amounts  of  Fixation  with  serum  of 

antigenic  serum  Man  Monkey  Goat 

0.01  +++ 

0.001 

o.oooi  ++4- 

O.OOOOI  -j- 

O.OOOOOI 
0.0 
12 


178  THE  PRINCIPLES  OF  IMMUNOLOGY 

The  above  protocol  shows  that  anti-human  serum  is  capable  of 
fixing  complement  in  the  presence  of  an  amount  of  antigenic  serum, 
which  is  considerably  less  in  the  case  of  human  antigenic  serum  than 
in  the  case  of  monkey  antigenic  serum.  Thus  the  group  reaction  is 
indicated  by  the  fact  that  the  serum  of  a  closely-related  species  is  in 
certain  doses  sufficient  to  produce  fixation.  Muir  and  Martin,  also, 
were  able  to  demonstrate  similar  reactions  in  several  different 
animal  groups. 

Relation  of  Complement-fixing  Bodies  to  Other  Immune 
Bodies. — The  fact  that  the  treatment  of  animals  with  a  protein  in 
solution  can  lead  to  the  development  in  the  animal's  serum  of  a  capacity 
both  for  precipitating  the  antigen  and  combining  with  the  antigen  to  fix 
complement  suggests  naturally  that  there  may  be  some  relationship 
between  the  two  phenomena.  Gay  and  Moreschi  independently  were 
able  to  show  that  precipitates  formed  by  the  action  of  a  specific  immune 
serum  can  so  bind  complement  as  to  prevent  its  action  upon  a  hemolytic 
system.  The  assumption  is  justified,  therefore,  that  the  two  phenomena 
are  very  closely  related  and  may  indicate  that  complement  fixation 
depends  in  part  at  least  upon  fixation  of  the  complement  by  a  precipitate. 
The  question  naturally  arises  then  whether  or  not  there  may  be  com- 
plement fixation  without  precipitation  or  precipitation  without  com- 
plement fixation.  Furthermore,  a  fundamental  problem  is  whether  or 
not  the  two  activities  of  the  antiserum  depend  upon  two  different 
immune  bodies  in  the  serum  or  upon  the  double  capacity  of  the  same 
immune  body.  Neisser  and  Sachs  were  able  to  show  that  complement 
fixation  occurred  with  very  much  smaller  amounts  of  antigen  than  does 
visible  precipitation.  As  has  been  mentioned  before,  in  reference  to  the 
delicacy  of  the  reaction,  it  was  pointed  out  that  fixation  of  complement 
may  occur  with  dilutions  of  1-1,000,000,000,  whereas  visible  precipita- 
tion has  never  occurred  in  such  marked  dilution  of  antigenic  or  of 
immune  serum.  Thus  it  can  be  concluded  that  the  presence  of  a  visible 
precipitate  is  not  necessary  for  the  fixation  of  complement,  a  statement 
amply  corroborated  by  Muir  and  Martin.  Wassermann  and  Bruck 
found  that  by  permitting  bacterial  extracts  to  stand  for  a  considerable 
time,  the  extracts  were  no  longer  precipitable  in  the  presence  of  specific 
precipitating  immune  sera,  whereas  fresh  extracts  show  beautiful  pre- 
cipitation. Nevertheless,  both  new  and  old  bacterial  extracts  were 
found  to  fix  complement  in  the  presence  of  the  specific  immune  serum. 
Liefmann  further  showed  that  the  action  of  heat  may  so  alter  the 
antigenic  protein  as  to  lead  to  differences  in  complement  fixation  and 
precipitation.  He  immunized  rabbits  with  egg-white  and  found  that 
after  heating  the  egg-white  it  could  be  so  changed  that  it  was  no  longer 
precipitable  by  the  immune  serum  but  could  still  operate  with  the 
immune  serum  in  complement  fixation. 

Felke  and  also  Garbat  have  found  that  anti-typhoid  vaccination  in 
man  leads  to  the  production  of  agglutinins,  but  to  no  or  very  slight 
production  of  complement-fixing  bodies.  Felke  found  that  in  the  course 
of  typhoid  fever  and  during  convalescence  complement  fixation  could 


COMPLEMENT  FIXATION  179 

be  demonstrated  in  addition  to  agglutination.  Most  of  the  preceding 
experiments  indicate  that  the  phenomena  of  precipitation  and  comple- 
ment fixation  are  not  necessarily  associated,  but,  on  the  other  hand, 
cannot  be  interpreted  to  indicate  that  the  immune  serum  contains  two 
different  immune  bodies.  Friedberger  and  Liefmann,  working  inde- 
pendently, showed,  however,  that  heating  an  immune  serum  to  67°  C. 
can  destroy  the  precipitin  in  the  serum  without  altering  the  capacity 
of  the  serum  for  participating  in  complement  fixation.  This  experiment 
has  been  interpreted  as  indicating  that  precipitating  and  complement- 
fixing  bodies  represent  independent  activities  but  not  necessarily  that 
they  are  different  bodies.  Muir  and  Martin  found  that  upon  immuniz- 
ing animals  they  were  able  to  demonstrate  that  the  serum  of  these 
animals  contained  complement-fixing'  powers  earlier  than  precipitins 
could  be  demonstrated.  Altmann  found  that  complement-fixation 
bodies  appeared  earlier  than  agglutinins  for  paratyphosus  B  and  colon 
bacilli  but  with  the  use  of  typhoid  bacilli  both  bodies  appeared  about 
the  same  time.  As  a  converse  of  this  demonstration,  Moreschi  im- 
munized birds  with  rabbit  serum  and  found  in  contravention  to  his 
earlier  work  that  he  was  able  to  produce  a  precipitin  of  very  high  titer 
without  being  able  to  demonstrate  the  power  of  complement  fixation  on 
the  part  of  the  immune  serum.  This  was  corroborated  by  Sobernheim. 
Liefmann  was  able  to  show  a  certain  amount  of  difference  in  the  activity 
of  immune  serum.  He  brought  the  immune  serum  in  contact  with  the 
antigen  at  o°  C.  for  sufficient  time  to  produce  a  considerable  amount 
of  precipitate.  He  then  centrifuged  the  precipitate  and  found  that  the 
supernatant  fluid  at  37°  C.  was  capable  of  fixing  complement.  Lebailly, 
by  the  fractional  addition  of  antigen  to  the  precipitating  immune 
serum,  was  able  apparently  to  separate  the  precipitating  and  comple- 
ment-fixing bodies.  Arlo  precipitated  the  antigenic  and  immune  sera 
by  means  of  CO2,  thereby  obtaining  the  globulins  in  the  precipitate. 
In  both  instances  the  complement-fixing  body  was  found  in  the  redis- 
solved  globulin  fraction  and  the  precipitating  body  was  found  in  the 
supernatant  fluid.  This  has  been  controverted  by  Bruynoghe,  who 
maintains  that  euglobulins  are  capable  of  producing  non-specific  fixa- 
tion. Reviewing  all  this  experimental  evidence,  it  seems  perfectly 
clear  that  complement  fixation  can  and  does  occur  independently  of 
visible  precipitation,  a  statement  supported  by  a  great  mass  of  more 
recent  investigation  of  the  subject.  None  of  these  experiments,  how- 
ever, can  be  safely  interpreted  as  indicating  that  there  are  two  separate 
bodies  in  the  immune  serum.  Neufeld  and  Handel,  however,  appear  to 
be  definitely  of  the  opinion  that  there  are  two  separate  bodies  concerned. 
They  found  that  sensitized  cholera  spirilla  are  capable  of  fixing  the 
hemolytic  complement  at  o°  C.  but  that  at  37°  C.  the  organisms  will 
fix  both  hemolytic  and  bacteriolytic  complement.  They  explain  this 
by  assuming  that  the  fixation  at  higher  temperature  is  due  to  the  bacteri- 
cidal amboceptor  but  that  the  fixation  at  o°  C.  is  due  to  a  separate  sub- 
stance which  they  named  the  Bordet  antibody.  Such  experimental 
evidence  cannot  be  accepted  as  final.  Sachs  states  that  a  priori  it  can 


i8o  THE  PRINCIPLES  OF  IMMUNOLOGY 

be  supposed  that  the  antigenic  protein  can  simultaneously  combine  with 
precipitins  and  with  amboceptor.  He  offers  the  hypothesis  that  one 
immune  molecule  may  contain  different  binding  complexes,  one,  for 
example,  combining  with  precipitins  to  produce  a  precipitate  and  the 
other  combining  with  the  antigen  and  the  complement  to  produce  fixa- 
tion. If  this  view  be  accepted,  the  experiment  of  Friedberger  and  Lief- 
mann,  in  which  the  immune  serum  was  heated,  indicates  that  of  the 
two  binding  complexes  the  precipitating  one  is  the  more  labile.  It  can 
very  readily  be  seen  that  the  interpretation  of  Sachs  depends  almost 
entirely  upon  an  acceptance  of  the  Ehrlich  hypothesis  of  the  structure 
of  immune  bodies. 

Dean  has  examined  the  question  and  finds  that  the  optimal  rela- 
tionship between  antigen  and  immune  serum  for  the  production  of 
precipitation  is  by  no  means  necessarily  the  optimal  relationship  for 
complement  fixation.  Therefore,  the  two  phenomena,  as  has  already 
been  pointed  out,  are  by  no  means  parallel.  He  is  of  the  opinion, 
however,  that  this  lack  of  parallelism  is  not  necessarily  an  indication 
that  the  two  things  are  entirely  distinct  and  separate.  He  is  of  the 
opinion  "  that  they  represent  two  phases  of  the  same  reaction."  The 
complement  fixation  represents  the  earliest  and  more  delicate  stage 
of  a  reaction  which,  in  its  more  marked  manifestation,  is  seen  by  the  for- 
mation of  a  precipitate.  Zinsser  has  studied  the  matter  carefully  and  has 
come  to  the  conclusion  "  that  the  precipitation  is  merely  a  secondary, 
colloidal  phenomenon,  which  may,  or  may  not,  coincide  with  the  phase 
of  greatest  alexin  (complement)  fixation,  according  to  other  fortuitous 
conditions  which  may  favor  or  retard  flocculation."  He  found  that  a 
mixture  of  sheep  serum  and  its  specific  immune  serum  showed  com- 
plement-fixing activity  only  in  the  precipitate.  On  the  other  hand,  in 
a  mixture  of  a  filtrate  of  typhoid  bacilli  and  a  specific  immune  serum 
both  the  precipitate  and  the  supernatant  fluid  were  capable  of  fixing 
complement.  "  From  this  it  seems  to  follow  that  immunization  with 
the  more  complex  cellular  elements  has  given  rise  to  the  precipitating 
antibodies  present  also  in  the  anti-sheep  serum,  and  in  addition  to 
this  to  sensitizers  which  are  not  precipitable  (remaining  in  the  super- 
natant liquid)  and  not  present  in  the  anti-sheep  serum."  He,  therefore, 
is  of  the  opinion  that  since  both  the  antigen  and  the  immune  body  are 
colloidal  in  character  they  may  be  expected  to  follow  the  laws  of 
colloids.  This  may  be  interpreted  to  indicate  that  the  contact  of  the 
mutually  precipitating  colloids  must  be  present  in  optimal  concentration 
in  order  to  show  a  visible  precipitation,  but,  on  the  other  hand,  .the 
interaction  of  the  two  bodies  which,  in  the  quantities  employed, 
show  no  visible  precipitate,  may  be  demonstrated  by  the  comple- 
ment-fixation test.  He  states  "  that  the  visible  precipitation  would 
seem,  therefore,  to  be  a  secondary  phenomenon,  the  essential  one 
being  the  union  of  an  antigen  with  a  sensitizer  by  which  it  is  ren- 
dered amenable  to  the  action  of  the  alexin  "  (complement). 

Is  the  Complement-fixing  Body  an  Amboceptor? — There  arises 
further  the  question  as  to  whether  or  not  the  body,  which,  in  combina- 


COMPLEMENT  FIXATION  181 

tion  with  antigen,  serves  to  fix  complement,  is  to  be  regarded  as  an 
amboceptor  (sensitizer).  As  has  been  shown  in  the  discussion  of 
cytolysins,  it  is  possible  at  o  °C.  to  bring  about  a  selective  combination 
of  hemolytic  amboceptor  with  its  antigen.  Liefmann  attempted  to 
bring  about  a  union  of  complement-fixing  body  and  its  antigen  in  this 
way  but  was  unsuccessful.  It  is  known  that  if  a  considerable  excess 
of  antigen  or  antiserum  is  present,  complement  may  also  be  absorbed 
at  o°  C.  and  in  such  an  experiment  as  Liefmann's  it  is  impossible  to 
say  that  such  an  excess  did  not  exist.  Therefore,  the  experiment  is 
not  conclusive.  Neufeld  and  Handel  also  attempted  selective  absorp- 
tion at  37°  C.  They  showed  that  cholera  vibrios  and  their  specific 
immune  sera  fix  the  hemolytic  complement  at  o°  C.,  whereas  the 
bacteriolytic  complement  remains  active.  At  37°  C.  both  complements 
are  fixed.  They  are  of  the  opinion  that  at  o°  C.  the  complement-fixing 
amboceptor  is  bound  to  the  hemolytic  complement  and  that  at  37°  C. 
both  the  complement-fixing  and  bacteriolytic  amboceptors  are  active. 
This  experiment  has  been  held  to  support  the  hypothesis  of  the  multi- 
plicity of  complements.  They  also  found  that  an  immune  serum  pro- 
duced by  the  injection  of  a  certain  water  vibrio  acted  as  a  complement- 
fixing  body  with  cholera  spirilla  but  did  not  serve  as  a  bacteriolytic 
amboceptor.  This  may  be  interpreted  as  indicating  that  the  two  im- 
mune bodies  are  distinct  but  does  not  prove  the  amboceptor  nature  of 
that  body  which  enters  into  the  phenomenon  of  complement  fixation. 
It  may  very  well  be  that  the  experimental  conditions  were  not  optimal 
to  the  reactions  and  that  while  investigators  sought  to  separate  two 
forms  of  complement  they  were  working  with  one  and  the  same  body 
which  operates  somewhat  differently  under  the  diverse  conditions. 
Sachs  interprets  the  amboceptor  as  a  body  which  brings  about  the  union 
between  antigen  and  complement  but  states  that  certain  amboceptors 
may  be  toxic  (lytic)  and  others,  for  example,  those  serving  to  fix  com- 
plement, may  be  considered  as  atoxic.  He  considers  that  the  differences 
in  effect  may  be  the  result  of  a  number  of  factors,  including  mass  action 
and  differences  in  combining  avidity  of  the  various  reacting  bodies. 
It  would  appear  to  us  that  Zinsser's  interpretation  in  regard  to  pre- 
cipitins  might  also  be  applied  here  and  that  the  lysis  of  cells  may  be  an 
incident  in  complement  fixation,  certain  conditions  favoring  lysis,  others 
merely  fixation  of  complement.  If  this  be  accepted,  the  complement- 
fixing  body  must  be  regarded  as  an  amboceptor  or  sensitizer  in  the 
same  sense  as  are  the  cytolysins. 

Activation  by  Complement. — -The  utilization  of  complement  in 
hemolysis  serves  so  to  fix  complement  that  it  cannot  activate  a  bac- 
teriolytic amboceptor.  Therefore,  hemolysis  exhibits  the  fixation  of 
complement  in  association  with  lysis  of  the  cells.  Handel  found  that 
hemolytic  and  complement-fixing  properties  of  an  immune  serum  were 
parallel,  but  Muir  and  Martin  observed  marked  differences.  The  latter 
investigators  produced  two  immune  sera,  one  against  ox  serum  and  the 
other  against  ox  cells,  both  of  which  exhibited  hemolytic  and  comple- 
ment-fixing properties.  The  immune  serum  prepared  against  ox  cells 


182  THE  PRINCIPLES  OF  IMMUNOLOGY 

laked  the  antigenic  cells  in  doses  of  0.0015  c.c.  and  fixed  complement  in 
the  presence  of  o.ooi  c.c.  ox  serum.  The  immune  serum  prepared 
against  ox  serum  hemolyzed  ox  cells  in  doses  of  0.05  c.c.  but  fixed 
complement  when  combined  with  only  0.000,001  c.c.  of  ox  serum.  The 
immune  serum  against  ox  serum  had  only  about  one-thirtieth  the 
hemolytic  power  of  the  immune  serum  prepared  against  ox  cells  but  was 
looo  times  more  powerful  in  fixing  complement.  They  found  that 
ox  cells  can  absorb  hemolysin  from  an  immune  serum  without  removing 
the  precipitating  or  complement-fixing  activity  and  conclude,  in  oppo- 
sition to  the  hypothesis  offered  at  the  end  of  the  preceding  paragraph, 
that  the  complement-fixing  body  and  the  hemolysin  are  distinct  and 
separate  immune  bodies. 

Fixation  of  the  Complement  of  Natural  Hemolysins. — In  the  case 
of  natural  hemolysins  the  complement  in  many  instances  is  apparently 
in  a  state  of  close  combination  with  the  thermostable  lytic  body.  The 
entrance  of  such  complements  into  the  phenomenon  of  complement 
fixation  has  only  rarely  been  demonstrated  and  then  only  in  the  case 
of  those  naturally  hemolytic  sera  in  which  it  is  possible  to  absorb 
hemolytic  amboceptor  at  o°  C.  without  at  the  same  time  removing 
the  complement. 

Nature  of  Antigen  and  Amboceptor. — The  chemical  character  of 
the  antigen  and  amboceptor  have  been  studied  more  particularly  in 
connection  with  investigations  of  the  Wassermann  test  and  will  be 
considered  in  the  discussion  of  that  application  of  complement  fixation. 
It  may  be  said  at  this  place,  however,  that  the  complement-fixing 
immune  body  will  resist  the  ordinary  inactivating  temperature  of  56°  C. 
and  is  therefore  to  be  regarded  as  thermostable  but  is  destroyed  by 
75°  C.  for  one  hour.  The  antigen  is  thermostable  in  the  same  sense 
but  is  reduced  in  activity  at  75°  C.  but  not  destroyed  until  100°  C. 
is  reached! 

Inhibition  of  Complement  other  than  by  Fixation. — Of  great  im- 
portance are  the  factors  that  exercise  an  influence  upon  complementary 
activity.  Those  which  operate  on  the  living  animal  have  been  discussed 
in  the  chapter  on  Cytolysins  (see  page  127).  There  was  also  presented 
a  brief  discussion  of  physical  conditions  such  as  heat,  exposure  to  light, 
desiccation,  etc.  All  these  factors  must  be  considered  in  interpretations 
of  complement  fixation,  and  in  addition  it  is  considered  desirable  to 
present  certain  other  conditions  which  may  be  gathered  into  three  classes 
(a)  chemicals,  (b)  various  tissues  and  fluids,  (c)  antisera. 

Anti-complementary  Chemical  Agencies. — The  salt  concentration 
of  the  media  for  complement  fixation  is  extremely  important  and  reaches 
its  optimum  at  a  point  isotonic  with  the  body  fluids.  The  action  of 
complement  is  decreased  in  hypotonic  and  absent  in  salt  free  media. 
Examination  of  this  phenomenon  leads  to  the  conclusion  that  such 
action  is  upon  complement  rather  than  upon  amboceptor,  and  Ferrata  is 
of  the  opinion  that  the  important  change  is  the  splitting  of  the  comple- 
ment into  mid-piece  and  end-piece.  Under  these  circumstances  the 
mid-piece  may  be  bound  to  the  amboceptor-antigen  complex,  but  as 


COMPLEMENT  FIXATION  183 

the  end-piece  remains  free,  complementary  activity  does  not  appear. 
This  explanation,  however,  is  only  hypothetical,  is  not  entirety  supported 
by  other  experiments  and  fails  to  take  into  account  the  influence  of  salts 
on  colloidal  suspensions  and  solutions.  Excesses  of  salts  also  interfere 
with  the  action  of  complement,  but  on  dilution  to  isotonicity  the  function 
is  immediately  restored.  Therefore,  the  salts  do  no  permanent  injury 
to  complement.  Hektoen  and  Reudiger,  as  well  as  Manwaring,  offer 
the  explanation  that  ionization  of  the  salt  permits  of  a  union  with  com- 
plement which  is  easily  reversible.  Certain  salts,  such  as  those  of 
bile  acids,  as  well  as  sodium  oleate,  permanently  injure  complement. 
The  salts  are  of  themselves  hemolytic,  but  serum  inhibits  their  hemo- 
lytic  activity.  The  amounts  which  are  hemolytic  in  themselves  com- 
pletely inhibit  complement  and  by  virtue  of  the  presence  of  serum 
cannot  produce  lysis. 

Acids  and  alkalis  in  considerable  concentration  permanently  destroy 
complement,  but  if  the  injury  be  due  to  a  dilute  alkali  the  comple- 
mentary activity  may  be  restored  by  neutralization.  It  appears  that 
moderate  concentrations  of  acids  destroy  complement  without  restora- 
tion by  neutralization.  Dilute  acids  accelerate  hemolysis  and  for  this 
reason  are  to  be  avoided  in  accurate  work  with  complement  fixation. 
Certain  protein  products,  such  as  urea  (also  urea  sulphate)  and  guani- 
din  are  anti-complementary. 

Colloids  may  also  inhibit  complement  as,  for  example,  the  organic 
colloids,  glycogen,  inulin,  pepton,  albumose,  gelatin,  etc.,  as  well  as 
inorganic  colloids,  such  as  quartz  sand,  kaolin  and  carbon.  Numerous 
indifferent  chemical  precipitates,  such  as  colloidal  iron  hydroxide  and 
protein  precipitates  inhibit  complementary  activity.  It  is  possible  that 
in  certain  measure  this  may  depend  upon  their  interference  with  the 
complement  amboceptor  and  antigen  behaving  as  interacting  colloids. 

The  influence  of  lipoids  on  complementary  activity  is  of  great  im- 
portance, particularly  in  the  Wassermann  test,  but  we  may  mention  at 
this  point  that  lecithin,  cholesterol,  protagon  and  tristearin  in  sufficient 
concentration  are  anti-complementary  as  well  as  certain  lipins,  including 
the  neutral  fats,  olive  oil,  triolein,  etc.  Added,  finally,  to  the  list  of 
chemical  agents  are  boric  acid,  benzoic  acid,  formalin,  sodium  fluoride, 
sodium  sulphite  and  extracts  of  certain  spices. 

Anti-complementary  Action  of  Cells,  Tissue  Extracts  and  Body 
Fluids. — As  was  pointed  out  by  von  Dungern,  most  animal  cells  either 
in  the  form  of  emulsions  or  cells  may  inhibit  the  activity  of  comple- 
ment. Muir  found  that  the  stroma  of  red  blood-corpuscles  enters  into 
fixed  combination  with  complement  and  that  if  washed  red  corpuscles 
are  heated  to  55°  C.  for  twenty-four  hours  they  also  will  combine 
directly.  The  union  does  not  take  place  at  o°  C.  but  occurs  readily  at 
37°  C.  The  combination  is  apparently  not  dissociable.  Not  only  animal 
cells  but  also  a  wide  variety  of  bacterial  emulsions  or  their  filtrates 
as  well  as  yeast  cells  fix  complement.  On  the  basis  of  the  Ehrlich 
hypothesis  this  may  be  due  to  the  union  of  complement  with  the  com- 
plementophile  groups  of  those  sessile  receptors  of  cells  which  by  im- 


184  THE  PRINCIPLES  OF  IMMUNOLOGY 

munization  are  overproduced  and  become  free  in  the  blood.  Other 
investigations,  particularly  those  of  Landsteiner  and  von  Eisler,  indicate 
that  the  cell  lipoids  play  a  part  in  the  union  with  complement. .  The 
material  extracted  from  the  cells  by  petroleum  ether  was  found  to  be 
definitely  anti-hemolytic  and  furthermore  this  was  especially  true  if 
the  cells  used  in  hemolysis  were  from  the  same  species  as  the  lipoidal 
extracts.  Landsteiner  and  von  Eisler  demonstrated  in  addition  that 
cells  treated  with  fat-dissolving  agents  were  less  susceptible  to 
hemolysis  than  normal  cells.  They  suggested  the  possibility  that  the 
fixing  substance  may  be  a  lipoid  protein  combination.  Bang  and 
Forssman  extracted  cells  with  ether  and  found  that  an  acetone  soluble 
material  could  be  recovered  that  was  definitely  anti-complementary. 
Dantivitz  and  Landsteiner  confirmed  this  but  found  in  addition  that  the 
fraction  remaining  in  the  ether,  the  acetone  insoluble  fraction,  could 
fix  normal  amboceptor  but  not  immune  amboceptors.  Thus  it  will  be 
seen  that  the  finer  details  of  the  anti-hemolytic  powers  of  lipoidal 
extracts  are  still  unsettled.  As  to  the  anti-complementary  action  of  bac- 
terial extracts  Zinsser  suggests  that  it  may  be  non-specific  and  com- 
parable to  the  anti-complementary  activity,  mentioned  in  the  previous 
paragraph,  of  such  inert  substances  as  kaolin  and  quartz  sand. 

The  body  fluids  of  importance  in  this  connection  are  the  tissue 
juices,  certain  pathological  exudates  and  more  particularly  the  blood 
serum.  Camus  and  Gley  found  that  a  normal  hemolysin  may  be  in- 
hibited by  the  addition  of  a  similar  serum  which  had  been  inactivated. 
Miiller  showed  that  a  heated  serum  may  inhibit  the  activity  of  other 
sera,  and  concluded  that  this  was  due  to  an  anti-complementary  activity. 
Extreme  instances  of  this  action  have  been  reported  by  Kenneway 
and  Wright.  Muir  and  Browning  demonstrated  that  inactivated  sera 
homologous  with  those  used  as  complement  were  more  strongly  anti- 
complementary  than  heterologous  sera.  They  concluded  that  the  action 
was  due  to  the  presence  in  the  heated  sera  of  complementoid  which, 
at  least  partly,  excluded  the  complement  from  union  with  the  ambo- 
ceptor. Bordet  and  Gay  found  that  a  sufficient  dilution  of  inactivated 
sera  removed  the  anti-complementary  action  and  therefore  consider 
concentration  of  the  serum  a  most  important  factor.  This  would  indi- 
cate that  the  inhibition  is,  in  general,  against  the  reaction,  although 
Sachs  offers  the  suggestion  that  the  dilution  provides  for  a  dissociation 
of  complement  and  anti-complement.  More  proof  than  is  now  at  hand 
is  necessary  in  order  to  admit  the  existence  of  an  anti-complement  in 
the  sense  in  which  Sachs  uses  the  term  Of  great  importance  is  the 
work  of  Noguchi,  who  found  that  whereas  heating  the  serum  to  56°  C. 
permits  of  the  demonstration  of  anti-lytic  powers,  a  temperature  of 
70°  C.  considerably  augments  this  activity.  Noguchi  was  able  to  extract 
from  both  serum  and  cells  by  means  of  ether  a  substance,  highly  ther- 
mostable (90°  C.),  which  exhibited  the  same  anti-lytic  properties 
as  the  serum.  The  removal  of  the  ether  extract  left  the  serum  free 
from  anti-lytic  activity.  He  named  the  substance  "  protectin  "  and 
believed  it  to  be  the  source  of  the  inhibiting  action  of  serum.  Noguchi's 


COMPLEMENT  FIXATION  185 

opinion  is  that  the  inhibiting  action  of  serum  is  largely  anti-com- 
plementary in  nature,  although  in  part  the  action  may  be  upon  the 
amboceptor.  The  great  thermoresistance  of  the  body  in  the  serum 
argues  against  the  assumption  of  an  anti-complement  in  the  strict 
immunological  sense.  The  action  may  well  be  anti-complementary, 
but  from  the  work  of  Bordet  and  Gay,  as  well  as  of  Noguchi,  it  would 
appear  that  the  concentration  of  colloids  associated  with  a  disturbance 
of  lipoidal  balance  or  combination  must  occupy  a  most  important  place 
in  hypotheses  concerning  this  phenomenon.  Of  practical  importance 
is  the  fact  that  prolonged  preservation  of  serum  increases  its  anti- 
lytic  capacity. 

Anti-hemolytic  Activity  of  Immune  Sera. — In  discussing  the  prop- 
erties of  complement  (see  page  137)  we  mentioned  the  experimental 
evidence  concerning  the  production  of  anti-lysins  and  anti-comple- 
ments by  the  injection  of  immune  and  normal  sera.  The  anti-lytic 
activity  of  such  immune  sera  was  thought  at  first  to  be  due  to  an 
anti-complement,  but  later  was  thought  to  be  the  result  of  action  upon 
the  amboceptor  or  sensitizer.  It  must  be  recognized,  however,  that  the 
injection  of  a  serum,  whether  it  contain  complement  or  immune  ambo- 
ceptor, leads  to  the  production  of  a  precipitin  and  that  such  precipitins 
can  be  demonstrated  in  the  immune  sera  containing  the  so-called  anti- 
complement.  As  has  been  pointed  out  in  the  preceding  discussion  on 
complement  fixation  the  presence  of  precipitates  serves  to  fix  com- 
plement and  this  probably  accounts  for  the  anti-lytic  and  anti-comple- 
mentary powers  of  the  immune  sera. 

The  fact  that  agglutination  of  the  red  cells  inhibits  their  lysis  was 
pointed  out  independently  by  Handel  and  by  Karsner  and  Pearce.  This 
renders  inadvisable  the  use  for  complement-fixation  tests  of  sera  which 
are  strongly  hemagglutinative. 


CHAPTER  IX 

APPLICATION  OF  COMPLEMENT  FIXATION  TO  THE 
DIAGNOSIS  OF  DISEASE 

THE  WASSERMANN  REACTION. 
INTRODUCTION. 
THE  ANTIGEN. 

NATURE  OF  THE  SYPHILITIC  ANTIGEN. 
THE    SYPHILITIC   "  AMBOCEPTOR." 

NATURE  OF  THE  SYPHILITIC  "  AMBOCEPTOR." 
THE  COMPLEMENT. 
THE   HEMOLYTIC   SYSTEM. 

PRESERVATION  OF  ERYTHROCYTES. 

INFLUENCE  OF  TEMPERATURE  UPON  THE  REACTION. 
TECH  NIC  OF  THE  TEST. 
THE  ANTIGEN. 
OTHER   REAGENTS. 
THE  TEST. 

MODIFICATIONS    OF    THE    TEST. 
SPECIFICITY  OF  THE  REACTION. 
DIAGNOSTIC  VALUE  OF  THE  REACTION. 
INTERPRETATION   OF  RESULTS. 
DEPENDABILITY  OF  THE  TEST. 
QUANTITATIVE  RESULTS  WITH  THE  TEST. 
TEST  OF  SPINAL  FLUID. 
POST-MORTEM    WASSERMANN   TESTS. 
COMPLEMENT    FIXATION    IN    TUBERCULOSIS. 

"  ACID  FAST  FIXATION." 

COMPLEMENT  FIXATION  IN  GONOCOCCUS  INFECTIONS. 
OTHER  COMPLEMENT-FIXATION  TESTS. 
GLANDERS. 
TYPHOID    FEVER. 
SMALLPOX. 
WHOOPING   COUGH. 
ECHINOCOCCUS  CYST. 
MALIGNANT  TUMORS. 
SPOROTRICHOSIS. 

THE  WASSERMANN  REACTION 

Introduction. — The  demonstration  of  complement  fixation  employs 
five  reagents,  syphilitic  antigen,  red  blood-cells,  syphilitic  serum,  hemo- 
lytic  serum  and  the  complement.  Having  any  four  of  these  known  it  is 
possible  to  determine  the  immunological  nature  of  an  unknown  fifth 
reagent.  This  unknown  may  be  an  antigenic  substance  or  may  be  an 
amboceptor.  In  the  forensic  tests  for  species  proteins  the  unknown  is 
the  questionable  protein  which  is  employed  as  an  antigen;  in  other 
tests  the  unknown  may  be  bacteria  or  bacterial  proteins.  In  the  Was- 
sermann  and  other  clinical  tests  the  unknown  is  an  amboceptor  or 
similar  substance,  produced  in  the  blood  and  other  body  fluids  of  the 
diseased  subject. 

After  preliminary  experiments  on  animals,  Wassermann,  Neisser, 
Bruck  and  Schucht  published  in  1906  the  results  of  a  series  of  com- 
plement-fixation tests  in  cases  of  human  syphilis  and  demonstrated  the 
186 


APPLICATION  OF  COMPLEMENT  FIXATION     187 

clinical  value  of  the  reaction.  The  widespread  use  of  the  reaction  has 
led  to  marked  advances  in  the  understanding  of  this  disease,  its  sequelae 
and  its  treatment.  This  application  of  the  Bordet-Gengou  phenomenon 
has  enabled  science  to  progress  far  toward  the  elimination  of  one  of 
the  greatest  plagues  of  mankind.  Wassermann  and  his  collaborators 
had  first  shown  that  the  Bordet-Gengou  phenomenon  was  applicable 
not  only  to  bacterial  suspensions  but  also  to  bacterial  extracts  and  from 
this  developed  the  proposition  that  the  causative  agent  of  syphilis 
might  act  as  an  antigen  in  extracts  from  syphilitic  organs.  The  test 
was  originally  performed  with  a  salt  solution  extract  of  the  liver  or 
spleen  of  a  syphilitic  fetus  (rich  in  treponema  pallidum),  inactivated 
human  serum,  guinea-pig  complement,  an  inactivated  hemolytic  im- 
mune serum  and  sheep  erythrocytes.  All  the  reagents  were  tested  and 
titrated  to  avoid  factors  of  error  and  proper  controls  were  instituted  in 
each  experiment.  Much  has  been  accomplished  by  further  study  in  the 
hands  of  numberless  investigators,  but  we  shall  limit  our  discussion 
to  those  features  which  are  of  fundamental  importance  in  the  under- 
standing and  application  of  the  test. 

The  Antigen.— The  preparation  of  the  antigen  is  one  of  the  most 
important  features  of  this  test.  It  would  be  supposed  that  an  extract 
of  a  pure  culture  of  the  treponema  pallidum  should  give  the  most 
specific  results.  This,  however,  has  not  proved  to  be  the  case.  It  is 
difficult  to  grow  the  organism  in  pure  culture  and  the  method  of  culti- 
vation interposes  difficulties  in  the  way  of  obtaining  pure  extracts. 
Results  are  variable  and  therefore  not  so  specific  as  with  the  use  of 
other  antigens.  Until  recently  the  organism  had  not  been  cultured  in 
vitro  and  Wassermann  and  many  of  his  successors  were  unable  to 
utilize  the  method.  Wassermann  selected  the  organs  of  syphilitic 
fetuses,  because  they  were  known  to  contain  large  numbers  of  tre- 
ponemata,  and  from  these  made  extracts  in  physiologic  salt  solution. 
He  cut  syphilitic  fetal  liver  in  fine  pieces  and  mixed  100  grams  liver 
with  360  c.c.  physiologic  salt  solution  and  40  c.c.  5  per  cent,  phenol 
solution.  This  was  shaken  for  twenty-four  hours,  centrifuged  and  the 
supernatant  fluid  employed  as  antigen.  Practical  experience  shows  that 
these  antigens  vary  considerably  in  strength  and  rapidly  lose  fixing 
power.  Deterioration  may  result  from  light,  air,  warmth  and  freezing, 
so  that  the  extract  must  be  kept  tightly  stoppered  in  the  dark  at  low 
but  not  freezing  temperature.  Marie  and  Levaditi  dried  and  pulverized 
the  liver  in  order  to  preserve  it  and  made  up  salt  solution  extracts  when 
needed.  Morgenroth  and  Stertz  preserved  the  organ  in  the  frozen 
state.  The  subsequent  work  of  Weil  and  of  Lansteiner  and  their  col- 
leagues indicated  that  tumor  extracts,  extracts  of  animal  tissues  and 
of  normal  human  tissues  would  operate  as  antigens.  More  recently 
Varney  and  Baeslack  have  employed  extracts  of  experimentally  inocu- 
lated testes  of  the  rabbit  in  that  stage  of  infection  when  the  organs  are 
richly  infiltrated  with  the  treponema. 

Landsteiner,  Miiller  and  Potzl  found  that  alcoholic  extracts  of 
guinea-pig  heart  serve  admirably  as  antigen.  Independently  Porges 


i88  THE  PRINCIPLES  OF  IMMUNOLOGY 

and  Meier  showed  that  alcoholic  extracts  of  normal  or  syphilitic  fetal 
organs  operate  equally  as  well  as  the  watery  extracts  of  syphilitic  or- 
gans. These  studies  demonstrated  that  the  antigen  in  the  Wassermann 
test  is  not  necessarily  derived  from  the  .treponema  pallidum,  is  alcohol 
soluble  and  therefore  is  largely  of  lipoidal  nature.  Landsteiner,  Muller 
and  Potzl  extracted  I.  gram  heart  with  50.  c.c.  absolute  alcohol  but 
this  method  has  been  somewhat  modified.  For  practical  purposes  50  c.c. 
absolute  alcohol  are  placed  in  a  wide-mouth  amber  bottle  and  as 
guinea-pigs  are  killed  in  the  laboratory  the  heart  is  freed  from  blood 
and  connective  tissue,  cut  into  a  few  pieces  and  placed  in  the  alcohol. 
When  ten  hearts  are  so  collected  they  are  dried  and  ground  in  a  mortar. 
Ten  grams  of  the  dried  powder  are  returned  to  the  alcohol  and  the 
volume  made  up  to  100.  c.c.  This  is  shaken  for  twelve  hours  and  placed 
either  at  60°  C.  for  about  twelve  hours  or  at  37°  C.  for  about  five  days. 
It  is  then  filtered  and  the  filtrate  preserved  in  a  cool,  dark  place.  Further, 
a  second  extraction  with  alcohol  of  the  first  dried  extract  yields  an 
antigen  of  greater  value  because  it  contains  less  lytic  and  anti-lytic 
substance,  although  it  may  be  slightly  weaker  in  fixing  power.  Appar- 
ently, however,  the  alcoholic  extracts  of  syphilitic  organs  produce  more 
specific  antigens.  To  prepare  such  an  antigen  100  grams  syphilitic 
liver  are  freed  from  surrounding  tissue,  washed  free  of  blood  and  cut 
into  fine  pieces-  This  is  extracted  in  1000  c.c.  absolute  alcohol  for  a 
week  at  37°  C.,  the  flask  being  shaken  several  times  daily.  It  is 
then  filtered  and  titrated. 

Porges  and  Meier  found  that  lecithin  could,  within  certain  limits, 
be  substituted  for  the  antigenic  extracts.  This  naturally  led  to  extensive 
investigation  of  the  nature  of  the  substance  or  substances  concerned. 
The  fact  that  ether  extracts  of  alcohol  soluble  antigen,  according  to 
Levaditi  and  Yamanouchi,  did  not  contain  antigen  led  to  the  thought 
that  salts  of  bile  acids  might  serve  as  antigens.  Neither  lecithin  nor 
salts  of  bile  acids  give  consistent  results  in  the  actual  test  and  at  the 
present  time  no  pure  substance  serves  well  as  antigen.  The  importance 
of  lecithin  was  further  emphasized  by  the  refined  technic  of  Noguchi 
in  preparing  the  so-called  acetone  insoluble  antigen.  This  method  ap- 
pears to  be  especially  adapted  to  the  use  of  normal  human  organs, 
particularly  heart.  The  tissue  is  cut  into  fine  pieces^jnixed  with  five 
times  its  weight  of  absolute  alcohol  and  placed  at  '37°  C.  for  from 
five  to  seven  days.  It  is  then  filtered  and  the  clear  filtrate  evaporated 
in  a  dish  by  means  of  an  electric  fan  or  in  a  vacuum  desiccator.  The 
residue  is  taken  up  in  as  small  a  volume  of  ether  as  will  permit  solution 
and  allowed  to  stand  overnight.  The  clear  supernatant  fluid  is  decanted 
and  slightly  evaporated.  To  it  is  added  four  volumes  of  acetone.  The 
supernatant  fluid  is  poured  off  and  the  precipitate  allowed  to  evaporate 
to  a  resinous  consistence.  Three-tenths  of  a  gram  of  this  mass  is 
added  to  a  mixture  of  i.o  c.c.  ether  and  9.0  c.c.  pure  absolute  methyl 
alcohol  and  preserved  in  a  dark,  cool  place.  According  to  certain  re- 
ports, it  would  appear  that  this  antigen  gives  positive  results  in  cases 
which  are  negative  with  other  antigens  and  in  which  syphilis  has  not 


APPLICATION  OF  COMPLEMENT  FIXATION      189 

been  demonstrated  by  clinical  examination.  It  is  useful,  how- 
ever, as  a  control  of  other  antigens  with  which  doubtful  results  have 
been  obtained. 

The  source  of  the  lecithin  appears  to  play  some  role  in  its  value  as  an 
antigen;  that  from  heart  is  most  active,  while  that  from  liver,  brain 
and  egg  yolk  follow  in  the  order  named.  An  extract  such  as  that 
recommended  by  Noguchi  contains  in  all  probability  a  mixture  of  lipoids 
and  unsaturated  fatty  acids;  Noguchi  and  Bronfenbrenner  found  the 
fixing  capacity  of  such  extracts  to  vary  in  accordance  with  the  content 
of  unsaturated  fatty  acids.  Browning  and  Cruikshank  found  that  the 
addition  of  cholesterol  to  the  antigen  augments  the  delicacy  of  the  re- 
action and  this  method  has  found  widespread  use  in  this  country, 
particularly  through  the  work  of  Walker  and  Swift.  The  latter  investi- 
gators recommend  the  addition  to  alcoholic  extracts  of  human  or 
guinea-pig  hearts  of  0.4  per  cent,  of  cholesterol.  In  the  hands  of  sev- 
eral workers  this  has  so  increased  the  fixing  power  of  the  antigen  as 
to  give  positive  results  in  the  presence  of  non-syphilitic  serum,  the 
so-called  false  positive  reactions,  and  with  the  development  of  the 
method  of  fixation  at  refrigerator  temperature,  to  be  described  subse- 
quently, it  has  been  discarded  in  several  laboratories.  Nevertheless,  the 
cholesterolized  antigens  are  found,  in  the  hands  of  numerous  workers, 
to  show  much  less  variation  in  fixing  capacity  than  the  non-cholesterol- 
ized  extracts  and  for  this  reason  are  recommended  for  routine 
laboratory  work. 

It  would  appear  that  the  antigenic  substance  in  the  Wassermann  test 
is  not  an  antigen  in  the  biological  sense,  for  it  can  be  obtained  from 
tissues  not  the  seat  of  a  syphilitic  infection  and  as  has  been  shown  by 
Fitzgerald  and  Leathes,  upon  injection  into  animals  it  does  not  lead  to 
the  formation  of  immune  substances. 

The  methods  of  preparing  syphilitic  antigens  have  been  multiplied 
in  great  number  and  cannot  be  included  in  the  scope  of  this  book.  Sim- 
plification of  preparation  has  been  attempted  with  variable  results. 
Of  interest  is  the  method  suggested  by  Ecker  and  Sasano.  They  quote 
Neymann  and  Gager  to  the  effect  that  primary  extraction  of  the  tissue 
with  ether  removes  substances  of  anti-complementary  power  but  only  a 
small  amount  of  the  lecithin.  Ecker  and  Sasano  suggest  three  ten- 
minute  extractions  with  ether  in  the  proportion  of  25.  grams  ground 
and  dried  heart  muscle  to  50.  c.c.  ether.  The  material  is  then  extracted 
for  one  hour  with  75.  c.c.  95  per  cent,  ethyl  alcohol  at  boiling  tempera- 
ture (78°  C.)  in  a  flask  connected  with  a  reflux  condenser.  An  antigen 
of  this  sort  has  retained  its  original  fixing  power  in  this  laboratory 
after  more  than  a  year. 

Nature  of  Syphilitic  Antigen. — Extracts  of  the  treponema  pallidum 
may  serve  as  antigens  and  are  true  antigens  in  the  biological  sense. 
Craig  and  Nichols,  however,  found  that  alcoholic  extracts  of  organ- 
isms closely  related  to  treponema  pallidum,  as  the  treponema  per- 
tenue  and  the  treponema  microdentium,  may  fix  complement  in  the 
presence  of  syphilitic  serum.  Extracts  of  animal  and  human 


190  THE  PRINCIPLES  OF  IMMUNOLOGY 

organs,  particularly  when  prepared  by  alcoholic  extraction  appear  to 
be  distinctly  more  dependable  than  treponema  extracts  in  the  reaction 
of  complement  fixation.  The  exact  nature  of  the  substances  in  the 
alcoholic  and  in  the  acetone  insoluble  extracts  is  not  definitely  known 
except  that  lecithin  constitutes  a  large  part  and  that  it  is  associated 
probably  with  other  lipoids  of  the  diaminophosphatid  group,  unsat- 
urated  fatty  acids  and  certain  proteins  or  protein  fractions.  That 
physical  conditions  are  of  importance  has  been  known  since  Wasser- 
mann's  early  work,  for  it  is  established  that  a  certain  degree  of  turbidity 
of  the  antigen  or  its  dilutions  is  necessary.  The  watery  extracts  are  in 
a  state  of  finely-suspended  colloidal  emulsion  and,  as  Reudiger  and 
others  have  pointed  out,  the  dilutions  of  the  alcoholic  or  acetone  insol- 
uble extracts  by  means  of  salt  solution  can  be  demonstrated  to  have  an 
optimal  degree  of  turbidity. 

The  Syphilitic  "Amboceptor." — This  is  contained  in  the  blood 
serum,  the  cerebro-spinal  fluid  and  other  juices  of  syphilitic  patients  and 
experimental  animals.  In  the  usual  technic  the  blood  serum  is  inac- 
tivated for  one-half  to  one  hour  at  56°  C.  in  order  to  remove  comple- 
ment, but  in  certain  modifications  of  the  test  the  serum  is  used  fresh  in 
order  to  utilize  human  complement  in  the  reaction.  Bronfenbrenner, 
Reudiger  and  others  have  shown  that  inactivation  of  the  serum  reduces 
its  fixing  power.  Bronfenbrenner  recommends  the  use  of  unheated 
serum  because  of  the  greater  delicacy  of  the  reaction.  In  this  way  it  is 
possible  to  use  for  the  test  0.04  c.c.  or  0.05  c.c.  serum,  instead  of  the 
usual  o.i  c.c.  With  such  small  amounts  of  serum  the  human  comple- 
ment is  a  negligible  factor.  Long  preservation  or  excessive  heating 
of  the  serum  may  render  it  anti-lytic  or  anti-complementary.  Con- 
tamination from  unclean  skin  and  glassware  may  make  it  either  anti- 
lytic  or  lytic.  The  ingestion  of  alcohol,  the  presence  of  bile  in  the 
blood  in  jaundice  or  fat  in  the  blood  after  a  heavy  meal  or  in  cases 
of  lipemia  may  all  interfere  with  the  activity  of  the  fixing  body  in  a 
syphilitic  serum;  sera  in  lipemia  may  be  markedly  anti-comple- 
mentary. There  has  been  much  discussion  of  the  fact  that  human 
serum  may  contain  natural  hemolysins  for  sheep  corpuscles.  In  such 
an  instance  the  corpuscles  may  be  dissolved  by  the  excess  of  ambo- 
ceptors  in  spite  of  slight  fixation  of  complement  by  the  syphilitic  ambo- 
ceptor,  thus  transforming  a  weakly  positive  into  a  negative  reaction. 
Sasano  has  found,  however,  that  the  use  of  an  excess  of  immune 
hemolytic  amboceptor,  for  example,  ten  to  twenty  units  and  one  and  one- 
half  units  of  complement,  determined  by  careful  titration  does  not  influ- 
ence the  result.  Thus  the  factor  of  hemolysis  by  the  normal  anti-sheep 
amboceptor  of  human  serum,  which  amboceptor  is  practically  never  pres- 
ent to  the  extent  of  more  than  four  units,  is  practically  negligible. 

The  serum  may  be  preserved  for  a  considerable  time  if  kept  cool  and 
in  sealed  ampoules  or  in  tightly-stoppered  bottles.  Reudiger  has  found 
that  mixing  equal  parts  of  fresh  inactivated  serum  and  pure  sterile 
glycerol  preserves  the  so-called  syphilitic  antibody  for  as  much  as 
two  years.  Under  these  circumstances  the  sera  may  become  anti- 


APPLICATION  OF  COMPLEMENT  FIXATION     191 

complementary,  but  this  property  can  be  removed  by  heating  to  56°  C. 
for  thirty  minutes,  and  the  sera  are  then  satisfactory  for  use.  He 
maintains  that  heated  glycerolated  sera  give  much  stronger  positive 
results  than  fresh  unheated  sera  and  somewhat  stronger  than  fresh 
heated  sera.  This  method  is  valuable  for  preserving  known  positive 
and  negative  sera  as  controls  for  the  Wassermann  test. 

Nature  of  the  Syphilitic  Amboceptor. — The  substance  in  the  blood 
which  acts  as  amboceptor  is  apparently  closely  related  to  the  globulins, 
especially  the  euglobulin.  Recently,  however,  Duhot  has  suggested  that 
the  albumin  is  of  importance.  Pfeifler,  Kober  and  Field,  as  well  as 
Rowe,  have  shown  an  increase  of  globulins  in  syphilitic  blood  and 
spinal  fluid.  Noguchi  has  taken  advantage  of  this  fact  in  his  butyric 
acid  test  of  spinal  fluid,  but  diseases  other  than  syphilis  may  lead  to 
increase  of  globulins  in  the  spinal  fluid.  Peritz  states  that  the  lipoid 
content  of  syphilitic  serum  is  increased,  but  Bauer  and  Skutezky  found 
no  parallel  between  lipoid  content  and  Wassermann  reaction.  Klaus- 
ner  believes  that  the  flocculent  precipitate  which  appears  on  addition 
of  0.6  c.c.  distilled  water  to  0.2  c.c.  fresh  syphilitic  serum  is  due  to  the 
high  lipoid  content  of  the  serum.  Weston  has  found  no  definite  increase 
of  serum  cholesterol  in  late  syphilis  and  no  parallelism  between  the 
serum  cholesterol  content  and  the  Wassermann  reaction. '  According 
to  Wells,  "  a  favorite  interpretation  of  the  Wassermann  reaction,  which 
seems  to  harmonize  with  the  facts,  is  that  there  is  a  precipitation  of 
serum  globulin  by  the  lipoidal  colloids  of  the  antigen  and  adsorption  of 
the  complement  by  this  precipitate."  This  is  supported  by  the  work 
of  Jacobsthal  who  has  demonstrated  such  precipitates  by  use  of  the 
ultra-microscope  even  when  they  are  invisible  to  the  naked  eye.  Holker 
has  recently  studied  the  colloidal  phenomena  and  finds  that  the  addi- 
tion of  antigen  to  syphilitic  sera  produces  a  turbidity  the  curve  of 
which  is  steeper  and  higher  than  with  normal  sera.  He  finds  that  the 
serum  is  an  emulsoid  and  the  antigen  a  suspensoid.  Salt  solution  dis- 
perses the  serum  and  precipitates  the  antigen,  thus  increasing  the  pro- 
tective state  of  the  serum.  Negative  sera  are  much  more  protective 
than  positive  sera  in  preventing  the  antigen  from  being  precipitated 
by  salt  solution. 

The  Complement. — As  has  been  pointed  out  in  the  general  discus- 
sion of  complement,  guinea-pig  complement  is  most  widely  useful  in 
immunological  work.  It  was  used  by  Wassermann  in  his  original  test 
and  is  extensively  employed  to-day.  From  the  practical  viewpoint  it 
has  certain  objections.  Animals  are  expensive  and  for  a  small  number 
of  tests  it  is  undesirable  to  sacrifice  an  animal.  This  objection  may 
be  overcome  by  bleeding  from  the  heart  or  from  an  ear  vein  (Rous), 
but  the  technic  of  both  these  operations  is  somewhat  difficult.  Owing 
to  the  lability  of  complement,  it  cannot  be  well  preserved  and  the  serum 
must  be  used  soon  after  collection.  The  use  of  dried  complement  in 
filter  paper  has  been  abandoned,  although  such  complement  papers 
may  be  preserved  for  a  few  weeks  in  vacuum  desiccators  or  in  tubes 
containing  calcium  chloride.  Drying  in  the  frozen  state  in  vacuo  has 


192 


THE  PRINCIPLES  OF  IMMUNOLOGY 


*l° 

l!l. 

aiis 

IS1-" 

|alS 


' 


i  I 


& 


ifil4ff    *  5     1 

1 1   li 


^S?  §§.«£»     3  3 

~,d£oooKS        :3    n2  Cqs 


^r 

so 


*  s 


.8    ^ 

Jl  1 


8    t! 


It 


O   <N    O 

gdS 


S 


•SlPlk 

.  NUUfs 


•J3  o 


•Sg>sfi5?s         s    ,3 

^--^     3  3 


11 


>i 


C/3 


«  '3 

s  a   *3     M 


° 


S  jp!f||* 

111 
ISIifa 

•SSSN 

Illllil 

<^ 


Sgg 


. 

-g 


°N 


•    S  . 
:    US 

:    8§ 

o      g-OT 

i  :i 

I  fi 

8     «  rt 


i 


SS 

O  w 

•s"« 

8.0} 


f| 

"S  ° 
< 


•O  -IH   C    rt   >         rj         C  "bO 

^    UnH  ^J    3  +J    .  C  0)'  = 


o  o 

•8.3 

y 


l|     a 

^    a 


& 


ii 


- 


S  3 

OT      W 


.a  I 
s  I 
l| 


APPLICATION  OF  COMPLEMENT  FIXATION     193 


£oJ 

tj2^j 

ill 

S.&3 

II! 


£i  "S'2'S 

3*511 
HJH 

^HH-^-d 

*.aa--s 


.IS 


63 


.  a  o     <y 

r(   crt   <-«   S.-H 


cS  0       .      rfo 


* 

tr  c 
o  c8 


8  1 


Hi6! 


-3  _ 
I  °'o 


i 


ffi        3 

8^JJ3« 
c  C-S  *> 
g'^-c  g-S 

C  e  c«  o  <" 

|l>Jn. 


J3  c  o 


194  THE  PRINCIPLES  OF  IMMUNOLOGY 

been  recommended  by  Shakell,  but  Karsner  and  Collins  found  that  the 
activity  was  lost  in  eleven  to  fifteen  days.  Moledzky  states  that  com- 
plement in  the  frozen  states  retains  its  strength  indefinitely,  but  Reu- 
diger  found  that  although  its  strength  is  somewhat  augmented  at  the 
end  of  one  week,  it  deteriorates  after  the  second  week  of  preservation. 
Preservation  for  even  these  periods  involves  a  good  laboratory  equip- 
ment and  considerable  skill.  Kolmer  recommends  the  addition  of 
chemically  pure  sodium  chloride  to  pooled  guinea-pig  sera  in  the  pro- 
portion of  0.425  gram  salt  to  10.  c.c.  serum.  This  is  effective  for  sev- 
eral weeks'  preservation,  and  dilution  is  so  adjusted  as  to  restore  the 
serum  to  practical  isotonicity.  Detre  used  rabbit  complement,  but  it  is 
not  as  desirable  as  guinea-pig  complement  and  has  the  same  objections. 
Human  complement  is  employed  in  several  modifications  of  the  Was- 
sermann  test,  but  is  present  in  human  serum  in  extremely  variable 
amounts  and  is  difficult  to  titrate.  It  is,  however,  easily  accessible, 
as  it  is  present  in  the  serum  to  be  tested  for  syphilitic  amboceptor. 
The  selection  of  the  complement  to  be  used  depends  to  a  certain 
extent  upon  the  hemolytic  system  and  the  modification  of  the  test 
which  is  employed. 

Complement  should  be  used  in  accurately-determined  amounts. 
Therefore,  titration  is  of  the  utmost  importance.  Guinea-pig  serum 
shows  individual  variation  in  complement,  but  in  large  laboratories 
this  may  be  in  part  overcome  by  the  "  pooling  "  or  mixing  of  the  sera 
from  several  guinea-pigs.  Such  pooling,  however,  does  not  remove  the 
necessity  for  titration.  In  some  laboratories  the  complement  is  diluted 
i  to  10,  and  the  hemolytic  amboceptor  titrated  each  day  by  testing. 
We  are  of  the  opinion  from  experience  and  in  view  of  the  work  of 
Sasano  that  the  complement  should  be  titrated  in  various  dilutions 
(see  page  190)  against  a  constant  amount  of  previously  titrated  hemo- 
lytic amboceptor.  In  either  case  the  titration  should  take  place  on  the 
same  day  as  the  Wassermann  tests  are  made.  The  use  of  a  single  unit 
of  complement  does  not  allow  for  the  presence  of  anti-lytic  bodies  in 
the  reagents  nor  for  possible  deterioration  of  complement.  At  37°  C. 
the  use  of  one  and  one-fourth  units  appears  to  be  most  satisfactory, 
whereas  at  ice-chest  temperature  the  use  of  two  units  appears  to  be 
more  desirable.  Titration  of  the  complement  should  be  most  accurate 
and  the  end-point  be  determined  only  by  absolutely  complete  hemolysis. 

The  Hemolytic  System. — Sheep  erythrocytes  and  the  correspond- 
ing hemolytic  immune  serum  obtained  from  the  rabbit  were  used  by 
Wassermann  and  are  widely  used  at  the  present  time.  Other  systems 
include  the  use  of  goat,  horse,  ox,  human  and  fowl  corpuscles,  with 
specific  antisera  obtained  by  immunizing  rabbits.  Certain  investi- 
gators have  depended  upon  the  normal  hemolysin  for  sheep  erythro- 
cytes often  found  in  human  serum.  This  is  a  variable  quantity  and 
almost  never  very  large.  Noguchi  has  summarized  the  hemolytic  sys- 
tems in  a  table  giving  all  the  essential  data  (see  pages  192,  193). 

The  preparation  of  a  hemolytic  immune  serum  has  been  discussed 
(see  page  117).  The  preservation  of  this  serum  in  the  moist  state  is 


APPLICATION  OF  COMPLEMENT  FIXATION     195 

highly  satisfactory  if  placed  in  amber  ampoules  in  a  refrigerator.  If 
considered  advisable  preservatives  may  be  added  such  as  0.5  per  cent, 
phenol  or  50  per  cent,  glycerol.  If  the  serum  is  of  high  titer  it  may 
be  preserved  by  desiccation,  particularly  if  frozen  and  dried  in  a  vacuum 
desiccator.  Noguchi  has  obtained  good  results  by  drying  the  serum  in 
filter  paper.  The  filter  paper  is  subsequently  cut  into  strips  and  titrated 
by  cutting  measured  lengths  of  the  strips.  We  have  found  that  this 
does  not  permit  of  sufficiently  accurate  titration  and  also  that  the  titer 
is  not  well  maintained. 

Preservation  of  Erythrocytes. — If  kept  in  a  cool  place  without  freezing, 
sheep  erythrocytes  show  slight  hemolysis  in  a  few  days  and  well-marked 
hemolysis  in  about  a  week.  The  cells  oi  other  animals  show  variable  degrees 
of  fragility,  those  of  the  dog  being  especially  fragile.  Various  methods  of 
preserving  sheep  erythrocytes  for  the  Wassermann  test  have  been  studied 
by  Reimann  in  this  laboratory.  The  methods  of  particular  value  are  preserva- 
tion with  formalin  (Bernstein  and  Kaliski)  and  with  the  solutions  of  Rous 
and  Turner.  For  formalization  the  sheep  blood  is  allowed  to  run  directly 
into  formalin  solution  in  the  proportion  of  0.5  c.c.  of  40  per  cent  formaldehyde 
solution  to  400  c.c.  blood.  The  blood  is  then  defibrinated  by  shaking  with  glass 
beads,  preserved  in  the  refrigerator  and  before  use  washed  three  times  with 
saline  for  use.  The  method  of  preservation  as  worked  out  by  Rous  and 
Turner  is  carried  out  in  the  following  manner:  The  sheep  is  bled  directly  into 
Locke's  solution  containing  i  per  cent,  sodium  citrate,  in  the  proportion  of 
i  part  of  blood  to  4  parts  of  solution.  The  corpuscles  are  separated  by  rapid 
centrifugalization  and  carefully  washed  three  times  in  Locke's  solution  containing 
0.25  per  cent,  gelatin.  The  cells  are  then  placed  in  ampoules  in  a  layer  not 
more  than  2  mm.  in  depth  and  covered  with  saccharose-Locke  solution  to  a 
depth  of  about  2  cm.;  the  ampoules  are  sealed  and  stored  at  a  temperature  of 
5°  C.  to  6°  C.  Just  prior  to  use  the  cells  are  washed  with  .85  per  cent,  saline 
to  remove  the  saccharose  solution,  and  proper  dilution  effected  with  saline. 
Strict  asepsis  is  to  be  observed. 

THE  SOLUTIONS 

Locke-sodium  citrate  solution : 

Sodium  citrate   10  grams 

Sodium  chloride    9.2  grams 

Sodium  bicarb 0.05  gram 

Potassium  chloride o.i  gram 

Calcium  cholride o.i  gram 

Aq.  dest.  q.s.  ad 1000  c.c. 

Locke-gelatin  solution: 

Gelatin  2.5  grams 

Sodium  chloride    9.2.  grams 

Sodium  bicarb .* 0.05  gram 

Potassium  chloride  o.i  gram 

Calcium  chloride o.i  gram 

Aq.  q.s.  ad 1000  c.c. 

The  Locke  and  saccharose  solutions  are  sterilized  separately  and  used  in 
the  proportion  of  2.8  c.c.  of  the  saccharose  solution  and  7.5  c.c,  of  the 
Locke's  solution. 

Saccharose  solution : 

Saccharose 103.0    grams 

Aq.  q.s.  ad 1000       c.c. 

Locke's  solution: 

Sodium  chloride    9.2    grams 

Sodium  bicarb 0.05  gram 

Potassium  chloride o.i     gram 

Calcium  chloride  o.i     gram 

Aq.  q.s.  ad 1000       c.c. 


196  THE  PRINCIPLES  OF  IMMUNOLOGY 

Reimann  found  that  the  cells  can  be  preserved  for  use  in  the  Wassermann 
test  for  3  to  4  weeks  by  formalization  and  for  21  to  25  days  by  the  Rous  and 
Turner  method.  "  The  readings  obtained  differ  from  those  obtained  with  fresh 
cells  only  in  so  far  as  some  s*era  produce  slightly  different  results  when  used 
with  cells  from  the  same  specimen  of  sheep  blood."  An  excellent  control  for  the 
usefulness  of  preserved  blood  is  suggested  by  Kolmer,  who  maintains  that  there 
should  be  no  discoloration  of  supernatant  fluid  after  the  second  washing  and 
that  the  blood  should  become  brighter  in  color  than  the  dark  color  it  possesses 
after  standing. 

When  extreme  accuracy  is  desired  cell  emulsions  are  made  to 
contain  1,000,000,000  cells  per  cubic  centimeter.  Such  emulsions  are 
being  more  widely  adopted,  but  many  laboratories  still  use  5  per 
cent,  or  10  per  cent,  emulsions  calculated  either  from  the  original 
blood  volume  or  the  bulk  of  the  centrifuged  cells.  The  cells  should 
always  be  most  carefully  washed,  so  as  to  avoid  precipitin  reactions 
which  may  appear  if  the  serum  is  not  entirely  removed  and  to  wash 
out  antilytic  substances  which  may  appear  if  the  blood  is  old. 

Influence  of  Temperature  upon  the  Reaction. — This  influence  may 
be  determined  as  regards  the  velocity  of  the  reaction  and  the  amount 
of  complement  fixed.  The  earlier  work  with  complement  fixation  was 
based  on  the  general  assumption  of  immunologists  that  a  temperature 
of  37°  C.  represents  the  optimum.  In  1912,  however,  A.  McNeil  pointed 
out  that  ice-chest  temperature  favors  the  completeness  of  complement 
fixation  in  the  Wassermann  test,  provided  the  time  of  exposure  is  from 
eight  to  twelve  hours.  This  was  confirmed  by  Coca  and  1'Esperance, 
Smith  and  W.  J.  McNeil,  Berghausen  and  others,  and  the  ice-chest 
method  has  now  been  adopted  by  a  large  number  of  laboratories  as  a 
standard  method.  The  time,  however,  has  been  reduced  to  from  three 
to  four  hours  and  the  results  appear  to  be  entirely  satisfactory.  The 
antigen,  .serum  to  be  tested  and  complement  are  mixed  and  placed  in 
the  ice-chest  for  the  required  time ;  the  mixture  is  then  brought  to  about 
37°  C.  in  a  water  bath,  the  sensitized  erythrocytes  added  and  the  whole 
incubated  at  37°  C.  for  one  hour. 

Dean  has  investigated  the  influence  of  temperature  and  finds  that 
fixation  proceeds  most  rapidly  at  37°  C.  Noguchi  confirms  this  but 
finds  that  at  the  lower  temperature  of  23°  C,  fixation  will  reach  a  maxi- 
mum but  proceeds  more  slowly.  He  states  that  with  the  acetone  insol- 
uble antigen  "  a  serum  containing  one  unit  of  fixing  substance  will 
complete  the  reaction  within  thirty  minutes  at  37°  C.,  sixty  minutes  at 
30°  C.,  and  two  hours  at  23°  C.,  irrespective  of  whether  human  or 
guinea-pig  complement  is  used."  Dean,  however,  finds  that  at  o°  C. 
the  amount  of  complement  fixed  is  much  greater  than  at  37°  C.,  and  this 
accords  with  experience  in  the  use  of  the  ice-chest  method.  Certain 
unknown  factors  may  delay  the  action  of  the  complement,  as  has  been 
pointed  out  by  McConnell,  and  a  second  incubation  may  accordingly 
have  to  be  prolonged  beyond  the  usual  time. 

The  Technic  of  the  Wassermann  Test. — For  the  demonstration  of  the 
method  we  may  use  an  alcoholic  extract  of  ox  heart  as  the  syphilitic  antigen, 
inactivated  human  serum  from  a  normal  individual  and  from  a  known  victim 
of  syphilis,  guinea-pig  complement  and  a  sheep  hemolytic  system. 


APPLICATION  OF  COMPLEMENT  FIXATION     197 


The  antigen  may  be  made  by  weighing  10  grams  ox  heart  which  has  been 
freed  from  blood,  fat  and  connective  tissue,  ground  in  a  meat  grinder  and  dried 
under  a  current  of  air  from  an  electric  fan  or  in  a  'desiccator.  It  is  then 
extracted  in  100  c.c.  95  per  cent,  ethyl  alcohol,  first  by  shaking  18  hours  in  an 
electrical  shaker  and  then  standing  for  5  days  at  37°  C.  It  is  filtered  and  kept 
tightly  stoppered  in  an  amber  glass  bottle  in  the  refrigerator.  For  use,  slowly 
add  9.0  c.c.  physiologic  salt  solution  to  i.o  c.c.  alcoholic  extract.  This  constitutes 
the  "antigen  dilution"  of  the  charts.  It  must  then  be  titrated  to  determine 
its  antilytic  properties  as  well  as  its  lytic  powers.  The  following  tests,  of  the 
antigen  may  be  set  up  after  previously  determining  the  titer  of  the  hemolytic 
amboceptor  and  complement.  In  the  following  titrations  the  complement  is 
diluted  so  that  i.o  c.c.  contains  I  unit,  the  amboceptor  so  that  i.o  c.c.  contains 
2  units.  In  the  first  series,  volume  is  made  up  by  addition  of  salt  solution,  so 
that  each  tube  contains  4.0  c.c.  and  in  the  second  series  so  that  each  tube 
contains  2.0  c.c. 

TlTRATION    OF    ANTIGEN    FOR    ANTILYTIC    PROPERTIES 


Antigen  dilution 

Complement 

1 

Hemolysin 

5  per  cent,  cell 
suspension 

| 

Result 

I.O  C.C. 

0.8  c.c. 
0.6  c.c. 
0.4  c.c. 

O.2  C.C. 

unit 
unit 
unit 
unit 
unit 
i  unit 

ncubate  one  ] 

2  units 
2  units 
2  units 
2  units 
2  units 
2  units 

I  C.C. 
I  C.C. 
I  C.C. 
I  C.C. 
I  C.C. 
I  C.C 

ncubate  one  ] 

P.H. 
P.H. 
P.H. 
C.H. 
C.H. 
C  H 

TlTRATION   OF   ANTIGEN  FOR   LYTIC   PROPERTIES 


Antigen  dilution 

5  per  cent,  cell  suspension 

IH 

rs 
o 

Result 

I.O  C.C. 

0.8  c.c. 
0.6  c.c. 
0.4  c.c. 

O.2  C.C. 

C.C. 
C.C. 
C.C. 
C.C. 
C.C. 
C.C. 

[ncubate  one  h 

P.H. 
P.H. 

In  the  protocols  P.H.  indicates  partial  hemolysis,  C.H.  complete  hemolysis 
and  ( — )  no  hemolysis.  Thus  it  is  seen  that  0.6  c.c.  is  the  smallest  amount  of 
antigen  which  is  antilytic  and  0.4  c.c.  the  largest  amount  which  is  not.  For 
practical  purposes  one-half  the  latter  amount,  or  0.2  c.c.,  is  the  largest  amount 
which  may  be  used.  This  is  considerably  smaller  than  the  amount  of  antigen 
which  possesses  hemolytic  properties  in  itself,  as  shown  in  the  second  protocol. 

After  obtaining  this  information,  the  antigen  should  be  titrated  to  determine 
the  smallest  dose  that  fixes  complement  in  the  presence  of  a  known  syphilitic 
serum.  A  strong  serum  (H — h++)  may  be  obtained  from  a  laboratory  or  if 
not  available  a  serum  may  be  secured  from  9.  patient  in  the  florid  secondary 
stage  of  the  disease.  This  serum  is  used  in  constant  amounts  of  0.2  c.c.  More 
delicate  titration  is  accomplished  by  the  use  of  a  known  ++  serum  either 
alone  or  in  addition  to  the  H — h+H~  serum.  Knowing1  that  0.2  c.c.  is  the  largest 
dose  of  antigen  dilution  that  may  be  employed  the  test  is  set  up  with  that  as  the 
maximum  amount  of  antigen,  followed  by  decreasing  doses.  (See  table  on  page  198.) 

The  protocol  includes  the  necessary  controls,  showing  that  neither  antigen 
(12),  syphilitic  serum  (7),  nor  non-syphilitic  serum  (19)  exhibits  antilytic 
powers.  It  shows  that  antigen  (13),  syphilitic  serum  (15)  and  non-syphilitic 
serum  produce  no  hemolysis.  It  shows  that  non-syphilitic  human  serum 
(15-18)  fails  in  the  presence  of  the  antigen  to  fix  complement.  It  shows  that 
in  the  presence  of  syphilitic;  serum  the  antigen  solution  in  amounts  as  small  as 
o.oi  c.c.  fixes  complement.  That  amount,  o.oi  c.c.,  is  the  fixing  dose  and  is 
doubled  for  the  actual  Wassermann  test. 


198  THE  PRINCIPLES  OF  IMMUNOLOGY 

TlTKATION  OF   ANTIGEN  FOR   FIXING   PROPERTY. 


Tube 

Antigen 
dilution 

Syphilitic 
serum 

Complement 

Hemolysin 

5%  Cell 
suspension 

Result 

O2        C  C 

O  2  n  C 

2  units 

2  units 

T    P  f* 

O  I        C  C 

O  2  C  C 

2  units 

2  units 

3 

4 

5 

O.OO5  c-c- 

0.2  C.C. 

2  units 

2  units 

C.C. 

P.  H. 

6 

O.OOI  C.C. 

0.2  C.C. 

2  units 

2  units 

C.C. 

C.  H. 

7 

O.2  C.C. 

2  units 

^2 

2  units 

C  C 

J3 

C  H 

8 

2  units 

o 

2  units 

C  C 

o 

P   Ii 

2  units 

C  C 

xo 

g 

C  C 

C 

ii 

2  units 

o 

C  C 

o 

12 

O.2        C.C. 

2  units 

g 

2  units 

C  C 

C  H 

1  1 

O  2  C  C 

^ 

ia 

14. 

O.2  C.C. 

i 

ICC 

8 

j-j 

r) 

Non  -syphili- 

HH 

tic  human 

serum 

15 

0.2        C.C. 

O.2  C.C. 

2  units 

2  units 

C.C. 

C.  H. 

16 

O.I        C.C. 

O.2  C.C. 

2  units 

2  units 

C.C. 

C.  H. 

i? 

0.05    c.c. 

O.2  C.C. 

2  units 

2  units 

C.C. 

C.  H. 

18 

O.OI      C.C. 

0.2  C.C. 

2  units 

2  units 

C.C. 

C.  H. 

19 

0.2  C.C. 

2  units 

2  units 

C.C. 

C.  H. 

20 

0.2  C.C. 



C.C. 



Other  Reagents  for  the  Test. — The  methods  of  obtaining  guinea-pig  blood, 
the  hemolytic  amboceptor  and  sheep  blood  have  been  described  (see  pages 
117  and  127).  Various  methods  are  in  vogue  for  obtaining  human  blood.  In  adults 
the  simplest  satisfactory  method  is  to  obtain  the  blood  by  puncture  of  one  of 
the  large  veins  in  the  cubital  fossa  anterior  to  the  elbow-joint.  The  needle  should 
have  a  calibre  of  about  i.  m.m.  and  although  sharp  should  not  have  an  elongated 
point.  The  fossa  is  cleansed  with  soap  and  water  followed  by  alcohol.  A  tourni- 
quet is  applied  at  the  middle  of  the  upper  arm  and  the  patient  instructed  to 
"  make  a  fist "  several  times  until  the  veins  stand  out  prominently.  The  sterile 
needle  is  inserted  and  the  blood  collected  in  amounts  of  5  to  10  c.c.  in  a  15  c.c. 
centrifuge  tube.  The  tourniquet  is  released  before  the  needle  is  withdrawn 
and  the  wound  sealed  with  collodion.  The  blood  is  allowed  to  clot,  the  clot 
separated  from  the  side  of  the  tube  by  means  of  a  sterile  needle,  and  allowed 
to  contract  for  several  hours  in  the  refrigerator.  The  tube  is  then  centrifuged 
and  the  serum  pipetted  into  another  tube  so  as  to  avoid  hemolysis.  The  serum 
is  inactivated  at  56°  to  6p°  C.  for  one-half  hour  before  testing,  unless  the  test 
is  to  be  made  by  a  modification  which  employs  human  complement.  Methods 
have  been  suggested  in  which  the  amount  of  blood  obtained  by  puncture  of  finger 
tip  or  ear  lobe  provides  sufficient  blood.  In  infants  or  obese  adults  blood  may  be 
obtained  by  the  use  of  a  scarifier  and  cupping.  Bleeding  from  the  longitudinal 
sinus,  from  the  great  toe  and  from  the  heel  are  also  practised  in  infants. 

The  Test. — With  the  reagents  at  hand  the  test  is  set  up  with  one  or  more 
antigens.  In  many  laboratories  different  types  of  antigen  are  employed,  as  for 
example  an  acetone  insoluble  antigen,  a  cholesterolized  alcoholic  extract  of 
heart  muscle  and  a  non-cholesterolized  alcoholic  extract  of  heart  muscle.  Others 
are  employed  as  the  operator  sees  fit.  Antigens  may  deteriorate,  so  that  it  is 
wise  to  have  several  on  hand  and  under  observation  in  the  test.  The  protocol 
shows  2  antigens  of  the  same  strength.  All  the  elements  in  the  test  are  to  be 
controlled  to  prove  that  they  are  not  antilytic  and  to  show  that  the  hemolytic 
system  operates  properly.  In  addition  it  is  essential  to  have  controls  with  a 
known  positive  and  a  known  negative  serum.  The  antigen,  complement  and 
hemolysins  are  diluted  so  that  the  proper  quantity  of  each  is  contained  in  i.q  c.c. 
It  appears  to  be  desirable  to  add  the  human  serum  without  dilution.  This  is 
done  with  a  i.o  c.c.  pipette  graduated  in  hundredths  of  a  cubic  centimeter. 
The  dotted  lines  in  the  body  of  the  protocol  indicate  that  salt  solution  is  to  be 
substituted  in  quantities  of  i.o  c.c. 


APPLICATION  OF  COMPLEMENT  FIXATION     199 

THE  WASSERMANN  TEST 


Tube 

Antigen 
No.  i 
dilution 

Antigen 
No.  2 
dilution 

Human  serum 

Comple- 
ment 

Hemolysin 

S  %   cell 
suspension 

Result 

I 

O.O2 

0.2 

2  units 

2  units 

C.C. 

2 

0.02 

O.I 

2  units 

2  units 

C.C. 

P.  H. 

3 



O.O2 

O.2 

2  units 

2  units 

C.C. 



4 

O.O2 

O.I 

2  units 

2  units 

C.C. 

P.  H. 

Known 

positive 

5 

O.O2 

O.2 

2  units 

2  units 

C.C. 



6 

O.O2 

O.I 

2  units 

2  units 

C.C. 



7 

O.2 

O.2 

2  units 

f-H 

3 

2  units 

C.C. 

f-H 

a 



8 

.... 

O.2 

O.I 

2  units 

1 

2  units 

C.C. 

0 

^ 



Known 

<u 

0 

negative 

8 

d 

o 

9 

O.O2 

.... 

0.2 

2  units 

<D 

2  units 

C.C. 

S 

C.  H. 

10 

O.O2 

O.I 

2  units 

1 

2  units 

C.C. 

j-i 

C.  H. 

ii 

O.O2 

O.2 

2  units 

vU 

2 

2  units 

C.C. 

3 

C.  H. 

12 

O.O2 

O.I 

2  units 

o 

c 

2  units 

C.C. 

o 
d 

C.  H. 

13 

O.O2 

2  units 

t—  < 

2  units 

C.C. 

1—  1 

C.  H. 

14 

O.O2 

. 

2  units 

2  units 

C.C. 

C.  H. 

15 

.... 

0.2  (test 

2  units 

2  units 

C.C. 

C.  H. 

serum) 

16 

0.2  (positive) 

2  units 

2  units 

C.C. 

C.  H. 

i? 

.... 

0.2  (negative) 

2  units 

2  units 

C.C. 

C.  H. 

18 

.... 

.  .  . 

2  units 

2  units 

C.C. 

C.  H. 

IQ 

211T1  li~C 

f\  f\ 

Ly 
2O 

.  .  . 



14.111  to 

C.L.. 

C.C. 

The  results  of  the  Wassermann  test  are  usually  indicated  by  plus  signs  ;  the 
following  diagram  indicates  the  interpretation  of  the  results: 


DEGREE  OF  HEMOLYSIS 
WITH 
0.2  c.c.  human  serum    o.i  c.c.  human  serum 


Result 


P.H. 


P.H. 
CH. 
C.H. 


++ 

+ 


In  these  readings  the  partial  hemolysis  is  relatively  small  in  amount.  If 
with  0.2  c.c.  human  serum  the  hemolysis  is  well  advanced  without  being  complete 
and  is  complete  with  o.i  c.c.  serum,  the  result  is  indicated  by  the  sign  +.  Other 
symbols  are  used,  but  the  results  are  indicated  in  the  same  general  way. 

Reference  to  the  protocol  shows  that  the  serum  in  tubes  I,  '2,  3,  4  is  positive 
for  syphilis  and  would  be  signified  as  a  three  plus  (H — h+)  serum.  The  known 
positive  is  a  four  plus  (++++)  and  the  known  negative  reacts  properly.  Tubes 
13  and  14  show  that  the  antigens  are  not  antilytic,  and  tubes  16,  17,  18  show  that 
the  sera  are  not  antilytic.  Tube  18  shows  that  the  hemolytic  system  operates 
properly.  Tube  19  shows  that  the  hemolysin  does  not  produce  hemolysis  with- 
out complement,  and  tube  20  shows  that  the  corpuscles  do  not  hemolyze  without 
the  other  agents. 

The  quantities  given  in  the  protocol  are  based  on  a  unit  of  i.o  c.c.  to  simplify 
the  explanation.  In  order  to  save  reagents  the  quantities  are  usually  divided  in 
half,  so  as  to  be  on  a  0.5  c.c.  basis.  The  directions  for  the  United  States  Army 
in  France  called  for  quarter  quantities,  so  as  to  save  reagents.  The  latter  direc- 
tions also  call  for  half  saturation  of  the  alcoholic  heart  extract  with  cholesterol 
(0.2  per  cent.).  Bronfenbrenner  has  suggested  the  use  of  o.i  c.c.  amounts  of  the 
reagents.  Methods  of  measuring  by  drops  have  been  employed,  but  are  inaccurate 
because  of  the  possible  variation  in  the  size  of  drops  unless  a  stalagmometer  or 
similar  instrument  is  employed. 


200  THE  PRINCIPLES  OF  IMMUNOLOGY 

Modifications  of  the  Tests. — Numerous  modifications  of  the  test 
have  been  recommended.  These  are  based  on  variations  in  syphilitic 
antigen,  various  ways  of  treating  the  human  serum,  differences  in 
selection  of  the  complement  and  in  selection  of  the  hemo- 
lytic  system.  These  are  indicated  in  the  chart  on  page  192.  It 
is  our  opinion  that  any  method,  to  be  acceptable,  must  per- 
mit of  accurate  measurement  of  the  reacting  bodies.  The  possi- 
bilities as  to  methods  of  preparing  antigen  and  human  serum  have  been 
discussed.  The  use  of  human  complement  in  the  test  interposes  errors, 
which  we  believe  have  not  been  overcome.  The  titration  of  human 
complement  must  differ  with  different  specimens  and  in  the  Gradwohl 
method  fails  to  take  account  of  the  variable  content  of  natural  hemo- 
lytic  amboceptor  in  human  serum.  Of  the  hemolytic  systems  recom- 
mended, the  most  satisfactory  are  the  sheep  or  goat  and  the  human 
systems.  In  most  laboratories  the  sheep  system  appears  to  be  most 
accessible  and  the  factor  of  error  introduced  by  the  presence  of  normal 
anti-sheep  amboceptors  in  human  serum  can  usually  be  overcome  by 
absorption  with  sheep  erythrocytes  or  can  be  controlled  by  the  use  of 
one  and  one-half  units  of  complement.  The  human  hemolytic  system 
largely  obviates  this  objection,  but  it  is  sometimes  difficult  to  obtain 
enough  blood  to  immunize  animals:  for  the  production  of  the  specific 
immune  hemolysin.  We  also  suggest  the  possibility  that  an  unusually 
strong  natural  iso-hemolysin  in  the  tested  serum  may  confuse  the 
results.  Kolmer,  in  a  recent  study,  has  found  that  the  human  hemo- 
lytic system  considerably  increases  the  delicacy  of  the  reaction,  espe- 
cially when  small  amounts  of  the  patients'  sera  are  employed.  In 
positive  cases  he  found  10  per  cent,  more  positive  reactions  by  the  use  of 
the  human  system  than  with  the  sheep  system. 

The  Specificity  of  the  Wassermann  Reaction. — Numerous  studies 
have  been  made  as  to  the  specificity  of  the  test  in  the  different  stages 
of  syphilis.  In  evaluating  such  figures  certain  factors  of  error  in  the 
actual  performance  of  the  test  must  be  considered.  Unless  the  worker 
is  familiar  with  the  many  factors  which  may  influence  the  reaction 
of  hemolysis  and  the  fixation  of  complement,  as  pointed  out  briefly 
in  the  chapter  on  hemolysis  and  the  discussion  of  complement  fixation, 
the  results  may  -be  misleading.  The  type  of  antigen  employed  is  also 
of  significance  as  influencing  the  results.  Of  no  small  importance  is 
the  operator  himself,  for  although  the  Wassermann  test  may  properly 
be  regarded  as  a  physico-chemical  test  rather  than  a  strictly  biological 
or  immunological  reaction,  nevertheless  it  requires  a  thorough  under- 
standing of  immunological  procedures.  Tests  made  in  the  hands  of 
persons  trained  to  perform  this  test,  without  broader  training,  are  not 
to  be  given  the  same  value  as  tests  in  the  hands  of  broadly-trained 
immunologists.  The  subject  of  specificity  of  the  test  is  closely  bound 
with  the  clinic,  in  which  certain  factors  of  error  in  clinical  diagnosis 
must  be  accepted.  Until  more  satisfactory  methods  are  provided  for  in 
the  post-mortem  room,  the  factor  of  error  there  is  almost  as  large  as 
in  the  clinic.  Warthin,  by  particularly  refined  methods  applied  to 


APPLICATION  OF  COMPLEMENT  FIXATION     201 

cases  which  have  been  examined  shortly  after  death,  has  shown  the 
presence  of  the  treponema  in  lesions  which  previously  had  not  been 
positively  known  to  be  syphilitic.  Symmers,  Darlington  and  Bittman 
found  a  considerable  divergence  between  ante-mortem  Wassermann 
tests  and  the  post-mortem  evidence  of  syphilis,  but  Turnbull  finds  a 
striking  agreement.  Certainly  syphilis  can  progress  for  a  long  time 
without  gross  morbid  anatomical  manifestations,  and  it  seems  possible 
that  the  pathologist  cannot  be  sure  of  excluding  syphilis  in  his  ana- 
tomical diagnosis.  Improved  technic  is  the  only  way  of  reducing  this 
factor  o>f  error  and  thereby  providing  an  accurate  control  of  the  Was- 
sermann and  other  clinical  tests. 

The  Diagnostic  Value  of  the  Wassermann  Test. — Naturally  this 
subject  has  been  studied  extensively  and  figures  vary  as  the  technic  is 
improved.  In  1914  Boas  published  an  analysis  of  over  8000  cases 
reported  in  the  older  literature  and  tabulates  them  as  follows : 

Number          positive      Per<*nt. 
of  cases  positive 

Primary  syphilis    1060  629  59 

Secondary  syphilis 3526  3181  90 

Tertiary  syphilis 1212  1020  84.1 

Early  latent  syphilis  983  504  51 

Late  latent  syphilis 1520  605  39 

Tabes  dorsalis 159  115  72 

Paresis 405  402  99.2 

These  figures  are  sufficient  to  indicate  that  the  Wassermann  test  is 
of  distinct  value  in  the  diagnosis  of  syphilis.  More  recent  statistics 
offered  by  Craig  as  the  result  of  tests  carried  out  by  himself  illustrate 
the  accuracy  of  the  reaction  as  applied  under  excellent  conditions. 
In  interpreting  the  following  figures  from  Craig,  given  as  the  result 
of  a  single  test  on  each  of  4658  cases  diagnosed  as  syphilis,  it  must 
be  remembered  that  there  is  at  least  a  small  factor  of  error  in  the 
clinical  diagnosis.  The  table  follows: 

Number          pnci>:w        Per  cent. 
of  cases          Positive         positive 

Primary  syphilis 908  813  89.5 

Secondary  syphilis 1889  1817  96.1 

Tertiary  syphilis 638  558  87.4 

Latent  syphilis 1 173  790  67.3 

Congenital  syphilis 28  25  89.2 

Parasyphilis   22  7  68.1 

4658  4010  86.2 

Tests  made  by  Craig  on  2643  individuals,  either  not  diseased  or  vic- 
tims of  disease  other  than  syphilis,  showed  the  reaction  to  be  positive  in 
eleven  instances  (0.4  per  cent.).  These  eleven  instances  included  four 
cases  of  malaria,  three  of  tuberculosis  (two  of  which  ultimately  gave  a 
clinical  history  of  syphilis),  three  cases  of  pityriasis  rosea  and  one  case 
in  which  the  diagnosis  was  not  established.  It  is  to  be  considered  pos- 
sible that  diseases  other  than  syphilis  may  produce  those  changes  in  the 
blood  which  lead  to  fixation  of  syphilitic  antigen  and  complement ; 
among  these  are  occasional  cases  of  leprosy,  scarlatina,  malaria,  try- 


202  THE  PRINCIPLES  OF  IMMUNOLOGY 

panosomiasis  and  certain  skin  diseases.  Gordon,  Thomson  and  Mills 
have  recently  insisted  that  malaria  will  not  produce  a  positive  re- 
action unless  complicated  by  syphilis  or  as  the  result  of  faulty  technic. 
Although  a  controversial  point,  we  believe  that  occasional  cases  of 
tuberculosis  may  give  a  positive  Wassermann  test.  That  this  is  not 
necessarily  due  to  coincident  infection  with  syphilis  is  shown  by  the 
experience  of  Petroff,  who  found  a  positive  Wassermann  in  a 
tuberculous  cow. 

Interpretation  of  Results. — Craig  and  others  are  of  the  opinion  that 
a  strongly  positive  result,  such  as  would  be  indicated  by  -| — | — | — |-  in 
our  schedule,  is  conclusive  evidence  of  syphilis,  whether  there  are 
symptoms  or  not.  Other  degrees  of  fixation  must  be  interpreted  with 
the  aid  of  clinical  history  and  symptoms.  A  single  negative  reaction 
does  not  exclude  syphilis.  In  doubtful  cases  the  so-called  provocative 
treatment  should  be  applied.  This  means  that  a  short  course  of  mer- 
cury or  preferably  half  the  usual  dose  of  salvarsan  or  neosalvarsan 
should  be  given  and  the  Wassermann  test  made  subsequently.  It  is 
advisable  to  test  the  blood  twelve,  and  twenty-four  hours  after  pro- 
vocative administration  of  salvarsan  as  well  as  every  day  for  at  least 
ten  days.  If  the  reaction  is  to  become  positive,  it  usually  does  so  in 
from  a  few  hours  to  five  or  six  days,  but  may  be  delayed  for  ten  days 
or  even  more.  That  this  is  an  absolutely  specific  effect  of  the  drug  is 
contradicted  by  the  report  of  Wildgren,  who  found  that  the  injection 
of  milk  may  produce  similar  results.  Endless  discussion  might  be  pre- 
sented as  to  the  interpretation  of  the  Wassermann  test  in  the  clinical 
diagnosis  of  syphilis,  but  we  incline  to  the  view  that  this  test,  as  is 
true  of  many  laboratory  examinations,  is  to  be  regarded  as  important 
evidence  in  clinical  diagnosis,  is  of  striking  specificity  when  properly 
performed,  but  is  not  absolutely  pathognomonic. 

Dependability  of  the  Test. — Criticism  has  been  directed  against 
the  test  because  of  the  fact  that  results  do  not  always  agree  with 
clinical  findings  and  because  of  differences  in  results  upon  the  same 
serum  in  different  laboratories.  It  must  be  admitted  that  the  factors 
of  error  in  the  test  are  greater  than  in  clinical  diagnosis  of  the  disease. 
Discrepancies  in  reports  from  different  laboratories  may,  in  part,  be 
due  to  inherent  faults  in  the  test,  to  faults  in  technic,  to  faults  in  selec- 
tion of  materials  and  to  insufficient  training  of  the  worker.  The  older 
literature  contains  serious  criticisms  of  the  test,  as  for  example  the 
papers  of  Wolbart  and  of  Uhle  and  Mackinney.  Under  the  direction 
of  the  Medical  Research  Committee  of  Great  Britain  in  1918  the  results 
obtained  independently  by  Dr.  C.  H.  Browning,  Dr.  J.  Mclntosh  and 
Col.  L.  W.  Harrison  upon  the  same  specimens  are  in  very  close  agree- 
ment. More  recently  Solomon  has  analyzed  the  results  of  3000  tests 
carried  out  in  two  different  laboratories  by  skilled  workers,  Dr.  Hinton 
and  Dr.  Castleman.  There  was  complete  agreement  of  results  in  9344 
per  cent,  of  this  large  series  of  tests.  This  study  demonstrates  that 
with  modern  methods  and  skillful  performance  of  the  test  results  are 
highly  dependable. 


APPLICATION  OF  COMPLEMENT  FIXATION     203 

Quantitative  Results  with  the  Wassermann  Test. — For  various 
purposes,  more  particularly  the  observation  of  the  results  of  treatment, 
it  may  be  desirable  to  titrate  accurately  the  amount  of  patient's  serum 
which  serves  as  an  amboceptor.  This  may  be  done  by  using  different 
quantities  of  the  serum.  Dilutions  of  the  serum  are  made  with  salt 
solution,  i  to  4,  i  to  8,  i  to  16,  i  to  32,  i  to  64,  or  are  measured  as 

o.i  c.c.,  0.05  c.c.,  0.03  c.c.,  0.02  c.c.,  o.oi  c.c.,  etc.  The  tubes  are  treated 

w 
in  the  usual  fashion  and  the  results  recorded  as-g-,  indicating  complete 

p 

fixation  in  dilution  I  to  8,  -^  indicating  partial  fixation  in  dilutions  of 

TT 

i  to  16,  — indicating  hemolysis  or  no  fixation  in  dilutions  of  i  to  32. 

O 

Wassermann  Test  on  Spinal  Fluid. — Spinal  fluids  are  not  inac- 
tivated and  are  employed  in  larger  volumes  than  blood  serum,  up  to  as 
much  as  i.o  c.c.  Hauptmann  and  Hossli  were  the  first  to  insist  upon 
the  use  of  large  quantities  of  spinal  fluid,  and  this  modification  changed 
the  entire  conception  of  the  frequency  of  positive  results  in  the  spinal 
fluid  of  such  diseases  as  paresis  and  tabes  dorsalis.  The  test  with  spinal 
fluid  is  of  particular  value  in  syphilis  of  the  central  nervous  system, 
where  it  is  somewhat  more  specific  than  the  test  with  blood  serum. 
The  test  has  also  been  used  with  success  with  transudates  and  exudates 
from  the  peritoneum,  pleura  and  pericardium.  Apparently  of  value 
in  examination  of  the  spinal  fluid  is  the  Lange  colloidal  gold  test, 
described  in  texts  of  clinical  pathology. 

Post-mortem  Wassermann  Tests. — In  a  certain  number  of  cases, 
death  ensues  too  soon  after  the  patient  comes  under  observation  to 
secure  blood  for  the  Wassermann  test.  Not  infrequently  the  result  of 
a  Wassermann  test  may  aid  the  pathologist  in  morbid  anatomical  diag- 
nosis and  may  furnish  information  of  value  to  the  clinician  in  the  con- 
sideration of  doubtful  cases.  The  question  arises  as  to  whether  or  not 
post-mortem  changes  in  the  blood  will  invalidate  the  Wassermann  test. 
Valuable  information  has  been  collected  by  Graves.  In  a  series  of 
400  cases  he  found  that  only  0.46  per  cent,  of  sera  from  cadavers  were 
antilytic  and  only  0.58  per  cent,  of  sera  were  hemolyzed,  coagulated 
or  otherwise  unfit  for  use.  The  post-mortem  and  ante-mortem  results 
were  the  same  in  97  per  cent,  of  sixty-eight  controlled  cases.  "  The 
reactions  conformed  to  the  anatomic  and  historical  evidence  in  304  of 
378  cases,  or  80.4  per  cent."  Contradictory  findings  are  recorded  in 
less  recent  literature,  but  we  believe  that  valuable  results  may  be  ob- 
tained with  blood  taken  after  death. 

COMPLEMENT  FIXATION  IN  TUBERCULOSIS 

The  advantage  of  a  complement-fixation  test  in  the  diagnosis  of 
early  pulmonary  tuberculosis  and  in  concealed  or  suspicious  lesions  is 
obvious,  Certain  authors,  Craig,  Miller,  von  Wedel,  report  a  high 
percentage  of  positive  reactions  in  tuberculous  individuals,  whereas 
others,  Cooper  and  Lange,  report  relatively  few  positive  results.  Petroff 
is  of  the  opinion  that  these  differences  may  be  due  to  lack  of  complete 


204  THE  PRINCIPLES  OF  IMMUNOLOGY 

and  careful  study  of  the  cases  clinically,  as  well  as  a  failure  to  observe 
minute  details  of  the  test. 

Antigens  are  of  the  utmost  importance,  and  numerous  forms  have 
been  suggested.  There  appears  to  be  well-founded  evidence  for  using 
several  strains  of  the  human  type  bacillus  associated  with  one  or  more 
strains  of  bovine  type.  The  methods  of  making  antigen  vary  and 
include  the  use  of  saline  suspensions  or  extracts  of  tubercle  bacilli, 
living  or  dead,  intact  or  pulverized ;  filtrates  of  broth  cultures ;  ether 
alcohol  extracts  of  whole  or  autolyzed  bacilli,  and  extracts  of  tubercu- 
lous organs.  Apparently  those  extracts  which  contain  both  lipoids  and 
proteins  are  most  satisfactory.  The  antigenic  substance  is  thermostable. 

The  human  serum  is  inactivated,  and  in  Petroff's  hands  appears  to 
be  most  satisfactory  if  collected  one  or  two  days  before  the  test. 
Accurate  titration  of  complement,  to  be  used  in  doses  of  two  units, 
and  of  hemolytic  amboceptor  is  essential.  Guinea-pig  complement  and 
a  sheep-rabbit  hemolytic  system  are  satisfactory.  It  is  absolutely  essen- 
tial that  glassware  be  perfectly  clean  and  that  measurements  be  accur- 
ate. The  incubation  of  the  mixture  of  antigen,  tuberculous  amboceptor 
and  complement  should  be  from  one  and  one-half  to  two  hours  at  the 
optimal  temperature  of  35°-4O°. 

Wilson,  using  a  lipoid-free  bacillary  antigen,  attaches  great  im- 
portance to  the  complement  and  finds  that  there  is  not  a  universal 
adaptability  of  guinea-pig  complement.  That  from  some  guinea-pigs 
appears  to  be  fixed  more  readily  than  that  from  others.  Therefore,  in 
general,  pooled  complements  are  likely  to  give  the  best  results.  If  a 
single  complement  is  used  tests  should  be  made  to  determine  the  extent 
of  fixation.  Von  Wedel  states  that  preservation  of  the  patient's  serum 
in  the  ice-box  for  five  to  seven  days  favors  the  reaction,  but  Petroff 
found  that  fresher  sera  are  preferable.  It  is  desirable  to  make  several 
tests  at  intervals  upon  the  same  patient.  As  the  result  of  1555  tests  on 
713  cases  Petroff  obtained  the  following  results: 

Cases  Positive    Negative   Percent. 

Clinically  active  tuberculous  212  199            13  93.9 

Quiescent  tuberculous  158  89            69  56.3 

Apparently  cured  more  than  two  years 58  5            53  8.5 

Normals    78  3            75  3.8 

Suspected    166  65  101  39.1 

Other  diseases 41  6            35  14.6 

An -analysis  of  these  figures  shows  that  under  proper  conditions 
complement  fixation  is  of  distinct  value  in  the  diagnosis  and  prognosis 
of  tuberculosis.  Basing  his  conclusions  on  experimental  data  Petroff 
considers  "  the  complement-fixation  test  in  tuberculosis  more  specific 
than  the  Wassermann  test "  in  syphilis,  an  opinion  in  which  we  concur. 
Nevertheless,  its  most  ardent  advocates  do  not  regard  the  test  as  pathog- 
nomonic  and  Petroff  regards  it  as  "  only  one  of  the  many  links  in  the 
tuberculosis  diagnostic  chain."  It  is  unfortunate  that  the  complement- 
fixation  test  gives  the  highest  percentage  of  positive  results  in  cases  in 
which  the  need  for  such  diagnostic  aid  is  least  evident,  namely  in  those 


APPLICATION  OF  COMPLEMENT  FIXATION     205 

cases  of  active  tuberculosis  in  which  the  diagnosis  on  clinical  and 
bacteriological  grounds  is  reasonably  certain. 

"Acid-Fast  Fixation." — Of  great  interest  is  the  fact  as  shown  by 
Cooke  and  others  that  the  complement-fixation  test  affirms  the  close 
biological  relationship  of  the  acid-fast  bacilli.  From  rabbits  immunized 
with  various  acid-fast  bacilli  Cooke  obtained  sera  which  reacted  inter- 
changeably with  each  member  of  the  group  employed  in  the  experiment. 
In  certain  instances  the  immune  sera  reacted  somewhat  more  strongly 
with  their  own  antigenic  organism  than  with  others  of  the  group.  Cooke 
also  found  that  the  sera  of  tuberculous  patients  react  not  only  with 
the  tubercle  bacillus  but  also  with  other  acid-fast  bacteria.  Sera  from 
cases  of  leprosy  also  contain  complement-binding  substances  which 
react  with  antigens  made  from  several  members  of  the  acid-fast  group, 
the  cases  of  nodular  leprosy  giving  more  striking  fixation  than  those 
of  anesthetic  type.  According  to  Cooke,  the  Wassermann  test  gives 
crossed  reactions  in  tuberculosis  which  are  too  frequent  to  be  ex- 
plained by  the  coincidence  of  syphilis. 

COMPLEMENT  FIXATION  IN  GONOCOCCUS  INFECTIONS 

As  with  other  immune  reactions  of  the  animal  body,  time  plays  an 
important  part  in  the  production  of  complement-fixing  bodies  in  gono- 
coccal  infections.  Acute  gonorrhea  is  usually  diagnosed  with  ease 
by  bacteriological  methods,  but  it  is  not  until  the  disease  has  persisted 
several  weeks  that  complement-fixing  bodies  are  likely  to  be  demon- 
strated. The  value  of  complement  fixation  appears  in  those  cases  where 
simpler  bacteriological  methods  are  not  adaptable,  such  as  gonorrheal 
rheumatism  and  endocarditis,  as  well  as  infections  of  deeper  parts  of 
the  genital  tract,  such  as  the  Fallopian  tube,  Cowper's  glands  and 
prostate.  The  test  is  also  useful  in  determining  the  cure  of  the  disease. 

Miiller  and  Oppenheim  in  1906  reported  favorable  results  with  the 
gonococcus  complement-fixation  test.  Bruck  and  subsequently 
Meakins  had  a  similar  experience,  but  more  recent  study  indicates 
that  the  older  methods  possess  little  specificity.  The  work  of  Teague 
and  Torrey,  Wollstein,  Watabiki  and  Schwartz  and  McNeil  demon- 
strated the  occurrence  of  numerous  immunologically  distinct  forms  of 
gonococcus  and  the  necessity  for  using  several  strains  in  the  antigen. 
It  now  appears  that  from  ten  to  fourteen  strains  are  desirable. 

The  production  of  antigen  has  been  extensively  studied.  Salt 
solution  extracts  appear  to  be  satisfactory.  Alcohol  extracts  have  no 
value,  and  Wilson  believes  that  a  lipoid-free  antigen  presents  an  im- 
provement in  titer,  stability  and  freedom  from  anti-complementary 
activity.  Warden  claims  good  results  with  an  antigen  composed  of 
salts  of  the  fats  of  the  gonococci.  Thomson,  working  under  the  direc- 
tion of  Col.  L.  W.  Harrison,  reports  excellent  results  by  dissolving 
the  organisms  in  decinormal  sodium  hydrate  solution  and  restoring 
to  the  neutral  point  by  decinormal  hydrochloric  acid.  In  the  hands  of 
most  workers  the  sheep-rabbit  hemolytic  system  appears  to  be  satis- 


206  THE  PRINCIPLES  OF  IMMUNOLOGY 

factory,  but  it  has  the  same  objections  as  obtain  in  the  Wassermann 
test.    A  human-rabbit  system  may  be  substituted  if  desired. 

The  test  appears  to  be  highly  specific  and  of  great  clinical  value 
when  properly  performed.  Although  the  gonococcus  and  meningo- 
coccus  are  closely-related  organisms  and  may,  according  to  Wollstein, 
give  crossed  complement-fixation  reactions  there  is  no  satisfactory 
evidence  that  epidemic  cerebrospinal  meningitis  in  man  produces  con- 
fusing complement-fixing  bodies.  Dixon  has  recently  studied  840 
tests  made  by  Dixon  and  Priestly  on  625  individuals.  Of  fifty-three 
strongly  positive  reactions  90.4  per  cent,  had  gonorrhea  or  a  history 
of  the  disease,  of  sixty-six  moderately  strong  reactions  86.3  per  cent, 
were  confirmed  clinically,  of  seventy-five  weakly  positive  reactions  72 
per  cent,  were  confirmed  clinically,  of  ninety  doubtful  reactions  58.9 
per  cent,  were  clinically  cases  of  gonorrhea.  Of  341  negative  reactions 
26.1  per  cent,  were  cases  of  gonorrhea  in  some  form;  of  these  only 
one  case  was  positive  to  a  second  test.  Therefore,  a  positive  test  is 
to  be  regarded  as  strong  presumptive  evidence  of  the  disease,  but 
both  positive  and  negative  reactions  should  be  controlled  by  sub- 
sequent tests. 

OTHER  COMPLEMENT-FIXATION  TESTS 

Glanders. — The  complement-fixation  test  in  this  disease  appears 
to  be  highly  specific  independently  of  the  strain  of  the  antigenic 
organism.  Its  principal  application  is  in  the  disease  as  it  affects  horses. 
The  mallein  test  and  the  agglutination  test  are  satisfactory  but  can  be 
supplemented  by  complement  fixation.  Occasionally  it  may  be  ser- 
viceable in  human  medicine. 

Typhoid  Fever. — Although  earlier  workers  obtained  variable  re- 
sults, later  investigations  in  the  hands  of  Garbat  and  of  Kolmer  with 
salt  solution  extracts  of  numerous  strains  of  the  bacillus,  the  so-called 
polyvalent  antigen,  have  given  excellent  results  more  particularly  in 
the  second  or  third  week  of  the  disease  or  later.  Blood  cultures,  the 
Widal  and  the  Dreyer  tests  are  so  much  more  easily  performed  that 
the  complement-fixation  test  is  to  be  regarded  as  only  supplementary. 
Nevertheless,  complement  fixation  is  more  likely  to  occur  in  the  course 
of  the  disease  than  as  the  result  of  prophylactic  vaccination  and  accord- 
ingly may  gain  diagnostic  value. 

Smallpox. — Positive  results  have  been  obtained  in  this  disease  by 
Jobling,  Sugai,  Dalm,  Klein,  Kolmer  and  others.  The  antigen  has  been 
obtained  either  from  the  lesions  of  vaccinia  in  calves  or  from  human 
smallpox  lesions.  Salt  solution  extracts  appear  to  be  better  than  alcoholic 
extracts.  In  addition  to  the  diagnostic  value,  the  reaction  adds  to  the 
evidence  concerning  the  biological  identity  of  smallpox  (variola)  vari- 
oloid  and  vaccinia.  Our  interpretation  of  Xylander's  results  indicates 
that  vaccinia  in  man  does  not  lead  to  the  establishment  of  complement- 
fixing  bodies  over  a  long  period  of  time  and  therefore  in  all  probability 
is  not  a  true  index  of  immunity  to  smallpox.  The  great  diagnostic 


APPLICATION  OF  COMPLEMENT  FIXATION     207 

value  lies  in  the  differentiation  of  smallpox  from  syphilis  and  from 
chicken-pox  (varicella). 

Whooping-Cough. — With  antigens  made  from  the  pertussis  bacil- 
lus of  Bordet-Gengou  the  reaction  appears  to  have  considerable  diag- 
nostic value. 

Echinococcus  Cyst. — The  antigen  is  obtained  by  filtering  the  cyst 
fluid  of  .man  or  sheep  and  preserving  with  0.5  per  cent,  phenol  in  a  cool 
place.  Varying  results  have  been  reported,  but  the  test  appears  to  be 
worthy  of  further  investigation  where  material  can  be  obtained  for 
its  use. 

Malignant  Tumors. — Numerous  attempts  have  been  made  to  aid 
in  the  diagnosis  of  malignant  tumors  by  the  complement-fixation  test, 
using  antigens  prepared  from  tumor  material.  The  results  have  been 
conflicting.  Von  Dungern  has  devised  a  test  using,  on  empirical 
grounds,  an  antigen  prepared  by  making  an  acetone  extract  of  normal 
human  red  blood-cells.  He  has  obtained  fixation  in  as  high  as  90  per 
cent,  of  known  cases  of  malignant  tumors.  The  test,  however,  has  not 
as  yet  been  sufficiently  widely  applied  to  justify  recommending  it  as 
of  clinical  value. 

Sporotrichosis. — Widal,  Abrami,  Joltrain  and  Weil  have  obtained 
excellent  results  using  as  antigen  the  sporotrichum  Beurmanni.  Moore 
and  Davis  have  recently  demonstrated  fixation  with  a  human  serum  in 
the  presence  of  Schenck-Hektoen,  Beurmann  and  Davis  strains  of 
the  organism.  This  reaction,  in  addition  to  agglutination,  is  of  distinct 
diagnostic  value. 


CHAPTER  X 
HYPERSUSCEPTIBILITY 

DEFINITION. 
OCCURRENCE. 

ANAPHYLAXIS. 

SENSmZATION. 
PERIOD  OF  INCUBATION. 
INTOXICATING  INJECTION. 
THE   REACTION. 

CLINICAL  PHENOMENA. 
DISTENTION  OF  LUNGS. 
FALL  IN   BLOOD-PRESSURE. 
METABOLIC  AND   BLOOD   CHANGES. 
DECREASED  COAGULABILITY  OF  BLOOD. 
DESENSITIZATION. 
PASSIVE   ANAPHYLAXIS. 
SPECIFICITY  OF   ANAPHYLAXIS. 
THEORIES   OF  REACTION. 

ANAPHYLACTIC  POISONS. 
CELLULAR  THEORIES. 
PHYSICAL  THEORIES. 
ANAPHYLACTOID   PHENOMENA. 
SUMMARY. 
THE  RELATION  OF  ANAPHYLAXIS  TO  IMMUNITY. 

Definition. — On  casual  consideration  hypersusceptibility  appears  to 
be  a  condition  exactly  the  opposite  of  immunity.  If  by  immunization 
an  animal  becomes  more  than  normally  resistant  to  a  poisonous  or  in- 
fective agent  so  in  the  state  of  hypersusceptibility  it  is  more  than  nor- 
mally susceptible  to  poisons,  to  infective  agents  and  to  agents  which  in 
the  normal  animal  appear  to  be  entirely  innocuous.  More  critical 
examination  of  the  phenomenon,  however,  has  led  to  the  conception 
that  hypersusceptibility  is  but  one  manifestation  of  the  intricate 
mechanism  of  immunity.  The  reasons  for  this  latter  conception  will 
appear  in  the  subsequent  discussion.  The  term  hypersusceptibility  is 
not  to  be  confused  with  anaphylaxis,  with  which,  in  our  judgment,  it 
is  not  synonymous.  We  prefer  to  limit  the  term  anaphylaxis  to  that 
state  of  hypersusceptibility  to  a  given  substance  which  has  been  induced 
by  a  previous  injection  of  the  same  substance.  The  reaction  is  limited 
to  proteins  or  protein  fractions.  Natural  hypersusceptibility  to  non- 
protein  substances  may  occur,  but  this  condition  cannot  be  induced  by 
a  previous  administration  of  such  substances. 

Occurrence. — Hypersusceptibility  may  be  natural  or  acquired. 
Undoubtedly  certain  individuals  in  whom  the  condition  is  supposed  to 
be  natural  have  acquired  the  state  by  preliminary  inoculation  of  the 
substance  to  which  they  are  susceptible.  This  may  be  an  unconscious, 
forgotten  or  concealed  acquisition.  The  introduction  of  practically  any 
protein  into  the  tissues  of  the  body  may  lead  to  the  acquisition  of  a 
hypersusceptibility  of  long  duration  unless  the  primary  inoculation  is 
succeeded  by  others  at  proper  intervals  and  in  proper  amounts  to  produce 
208 


HYPERSUSCEPTIBILITY  209 

immunity.  In  man  natural  hypersusceptibilities  are  believed  to  be 
manifested  upon  the  introduction  of  the  special  proteins  or  similar  sub- 
stances into  the  respiratory  tract,  the  alimentary  canal,  into  the  skin  and 
by  injection  into  the  tissues,  body  spaces  or  circulation.  Man  may 
exhibit  respiratory  symptoms  in  the  presence  of  vegetable  effluvia,  as  in 
"  hay  fever,"  "  rose  fever,"  and  of  the  effluvia  of  certain  animals,  such 
as  the  horse  and  guinea-pig1.  In  individuals  thus  susceptible,  local  or 
general  reactions  may  occur  following  inoculation  with  the  specific 
animal  or  vegetable  protein.  The  ingestion  of  animal  proteins,  such  as 
egg,  or  vegetable  proteins,  such  as  strawberry,  may  produce  severe 
gastro-intestinal  disturbances  sometimes  accompanied  by  general  symp- 
toms. In  certain  cases  this  hypersusceptibility  may  have  been  acquired 
by  previous  sensitization,  but  in  the  greater  number  no  such  explanation 
is  to  be  offered.  In  babies  susceptible  to  egg-white  there  is  no  prob- 
ability that  preliminary  direct  sensitization  occurs,  but  it  is  possible 
that  the  tendency  to  hypersusceptibility  may  have  been  inherited.  The 
instances  mentioned  are  examples  of  individual  hypersusceptibility. 
Although  less  clear  cut  there  are  also  evidences  of  species  hypersuscepti- 
bility, as,  for  example,  the  fact  that  ox  serum  is  distinctly  toxic  for 
guinea-pigs  and  much  less  so  for  man.  The  acquired  forms  of 
hypersusceptibility  will  be  considered  under  the  general  discussion 
of  anaphylaxis. 

Anaphylaxis. — Following  the  introduction  of  the  serum  treatment 
of  disease,  disturbing  elements  appeared,  the  most  striking  of  which 
were  the  frequent  production  of  "  serum  rashes  "  and  the  reports  of 
occasional  severe  constitutional  reactions  and  even  sudden  death.  Von 
Pirquet  and  Schick  pointed  out  on  the  basis  of  a  clinical  investigation 
in  connection  with  the  serum  treatment  of  diphtheria  and  scarlatina, 
that  in  from  seven  to  twelve  days  following  a  single  injection  of  serum 
or  several  injections  on  successive  days,  a  so-called  "  serum  disease  " 
appears.  This  is  characterized  by  macular  or  maculo-papular  eruptions 
of  urticarial  type,  malaise,  fever  and  other  symptoms.  After  this  period 
a  subsequent  injection  of  the  same  protein  leads  to  the  appearance  of 
similar  symptoms  and  signs  usually  within  twenty-four  hours.  After 
the  lapse  of  months  or  years  the  reaction  may  be  delayed  and  fail  to 
appear  for  several  days,  but  is  only  rarely  as  late  as  that  following  the 
primary  injection.  In  otfrer  words,  the  patients  appeared  to  have  been 
sensitized  by  the  primary  injection.  Not  being  able  to  define  exactly 
the  nature  of  this  condition,  the  name  allergy  was  suggested,  indicating 
an  "  altered  state  "  of  the  animal  body.  The  usage  of  the  term  at  the 
present  time  is  confusing  and  definitions  vary ;  we,  therefore,  prefer  not 
to  employ  it. 

Experimentally  similar  phenomena  had  been  noted  in  the  course  of 
other  studies  as  far  back  as  Magendie  in  1839,  but  it  remained  for 
Richet  and  Portier  in  1902  to  point  out  the  fact  that  an  animal  may  be 
rendered  hypersusceptible  to  a  poison,  by  the  previous  injection  of  a 
small  dose.  They  used  actino-congestine,  a  toxic  protein  extracted 
from  the  tentacles  of  actinia.  Because  the  phenomenon  indicates  a 
14 


210  THE  PRINCIPLES  OF  IMMUNOLOGY 

condition  the  opposite  of  prophylaxis  they  named  it  anaphylaxis.  As 
a  result  of  the  reports  of  accidents  following  the  use  of  diphtheria 
antitoxin,  Rosenau  and  Anderson  investigated  the  problem  experi- 
mentally and  found  that  the  danger  lies  in  the  serum  rather  than  in  its 
content  of  antitoxin.  They  demonstrated  that  the  reaction  is  specific 
for  the  protein  employed,  that  the  period  of  "  incubation  "  is  about  six 
days  and  that  once  established  the  sensitive  state  persists  for  many 
months  with  but  slight  reduction  in  intensity.  In  the  same  year,  1906, 
Otto  entirely  independently  published  similar  findings  in  Ehrlich's 
laboratory  as  the  result  of  an  interview  between  Ehrlich  and  Theobald 
Smith.  Smith  had  noted  that  animals  used  for  the  titration  of  diph- 
theria antitoxin  were  subsequently  extremely  sensitive  to  horse  serum. 
Otto,  accordingly,  employed  the  name  Theobald  Smith  phenomenon. 
Of  somewhat  similar  significance,  but  for  the  time  without  the  same 
direct  application  to  medicine,  were  the  investigations  of  Arthus,  who 
in  1903  published  a  study  in  which  he  showed  that  if  repeated  sub- 
cutaneous injections  of  protein  are  given,  the  fourth  and  subsequent 
injections  may  lead  to  severe  local  reactions  which  may  go  on  to 
gangrene.  If  a  later  injection  is  given  intravenously  death  may  result. 
Arthus  also  recognized  the  specificity  of  the  reaction.  The  year  1906 
marked  the  beginning  of  a  period  of  widespread  investigation 
of  anaphylaxis.  Much  has  been  learned  in  regard  to  the  mechanism 
of  the  process,  but  the  fundamental  principles  are  still  in  the  form 
of  hypotheses. 

The  Sensitization. — The  substances  necessary  for  the  demonstra- 
tion of  anaphylaxis  are  proteins.  These  need  not  contain  all  the  amino- 
acids,  for  Wells  has  shown  that  certain  vegetable  proteins,  zein,  hordein, 
gliadin,  lacking  "  one  or  more  such  amino-acids  as  glycocoll,  tryptophane 
or  leucine  produce  typical  reactions  "  and  Abderhalden  claims  to  have 
demonstrated  anaphylaxis  with  a  compound  polypeptid  made  up  of  four- 
teen amino-acid  molecules,  which  include  only  two  of  the  amino-acids, 
leucine  and  glycocoll.  The  sensitizing  substance  is  extremely  thermo- 
resistant.  Wells  finds  that  proteins  such  as  casein  and  ovO-mucin 
which  are  not  heat  coagulable  are  active  after  heating  to  100°  C.  and 
Besredka  has  found  that  if  a  coagulable  protein  is  so  diluted  as  to 
prevent  coagulation  it  withstands  temperatures  up  to  120°  C.  Rosenau 
and  Anderson  found  that  if  the  protein  be  in  the  dry  state  it  may  be 
heated  to  170°  C.  for  ten  minutes  and  upon  re-solution  will  serve  for 
the  production  of  anaphylaxis.  Heat  or  chemical  agents  which  render 
the  protein  insoluble  destroy  its  sensitizing  properties.  Trypsin  diges- 
tion has  the  same  effect.  Gay  and  Adler  reported  that  upon  frac- 
tioning  serum  with  ammonium  sulphate  the  euglobulin  contains  the 
sensitizing  substance,  but  not  that  substance  which  intoxicates  at  the  sec- 
ond injection.  Kato,  however,  finds  that  the  globulins  possess  the  largest 
content  of  both  sensitizing  and  intoxicating  substances.  Bogolomez 
and  subsequently  Meyer  claimed  that  anaphylaxis  could  be  produced 
with  lipoids  but  this  has  failed  of  confirmation  in  the  hands  of  Wilson 
and  of  White  and  others ;  it  is  not  generally  accepted.  The  chief  dif- 


HYPERSUSCEPTIBILITY  211 

ficulty  in  the  work  with  lipoids  lies  in  the  fact  that  it  is  practically 
impossible  to  obtain  the  lipoids  in  pure  form ;  an  extremely  small  amount 
of  adsorbed  protein  may  produce  the  reaction. 

The  method  of  sensitization  is  by  parenteral  routes,  although 
Rosenau  and  Anderson  in  their  original  communication  state  that  they 
had  been  able  to  sensitize  guinea-pigs  by  feeding  horse  serum.  Bes- 
redka  was  unable  to  confirm  this,  but  in  a  few  dogs  we  have  obtained 
results  which  have  been  highly  suggestive.  A  question  of  fundamen- 
tal importance  in  this  connection  is  whether  or  not  proteins  may  be 
absorbed  through  an  intact  intestinal  mucosa  without  digestion.  Ac- 
cording to  the  work  of  Van  Alstyne  and  Grant,  such  absorption  may 
occur.  Absorption  of  the  whole  protein  through  the  intestinal  mucosa 
or  the  mucosa  of  other  surfaces  might  well  serve  to  sensitize  animals 
or  man,  but  as  yet  the  problem  is  not  conclusively  settled.  If 
the  inoculation  be  by  parenteral  routes  there  is  apparently  little 
difference  in  outcome  whether  administered  subcutaneously,  intra- 
venously or  intraperitoneally. 

The  amount  of  protein  necessary  for  sensitization  is  extremely 
small.  In  their  original  work,  Rosenau  and  Anderson  found  that  in 
guinea-pigs  0.000,001  c.c.  horse  serum  suffices.  Wells  succeeded  in 
sensitizing  guinea-pigs  with  0.000,000,05  gram  crystallized  egg  albumin. 
Larger  sensitizing  doses  are  necessary  in  order  to  produce  subsequent 
death  from  anaphylactic  shock.  Such  minute  doses  are  not  applicable 
in  the  case  of  rabbits,  dogs  and  monkeys,  in  which  it  may  be  necessary 
to  inject  the  material  on  two  or  three  successive  days  in  order  to 
sensitize.  The  minimal  sensitizing  dose  in  man  is  not  known.  In 
experimental  animals  there  is  an  optimal  sensitizing  dose  which  bears 
a  certain  relation  to  the  subsequent  intoxicating  dose,  as  has  been  shown 
by  White  and  Avery.  In  a  general  way,  the  smaller  the  sensitizing 
dose,  the  larger  the  minimum  intoxicating  dose  and  vice  versa,  but  a 
sensitizing  dose  may  be  too  large  for  satisfactory  sensitization.  Accord- 
ing to  Besredka  the  larger  doses  also  require  a  longer  time  for  sensi- 
tization to  appear. 

Period  of  Incubation. — For  a  period  of  eight  to  twelve  days  after 
the  sensitizing  dose,  subsequent  injections  of  the  same  material  produce 
no  evidence  of  hypersusceptibility.  Gay  and  Adler  reported  that  if  the 
euglobulins  of  serum  are  employed  for  sensitizing,  the  period  of  incu- 
bation may  be  shortened  to  four  or  five  days.  If  during  this  period 
a  second  injection  be  given  the  animal  is  more  likely  to  become  immune 
than  hypersusceptible.  Rosenau  and  Anderson  found  that  the  state 
of  hypersusceptibility  increases  until  the  twenty-first  day,  after  which 
it  very  gradually  diminishes  but  persists  in  modified  form  probably 
throughout  the  life  of  the  animal. 

Intoxicating  Injection. — The  French  term,  injection  dechainante, 
is  highly  descriptive  of  this  part  of  the  process,  as  it  indicates  the 
explosive  character  of  the  manifestations  that  are  likely  to  occur.  This 
injection  may  be  intravenous,  intrameningeal,  intraperitoneal  or  sub- 
cutaneous. The  rapidity  of  reaction  and  severity  of  symptoms  are  most 


212  THE  PRINCIPLES  OF  IMMUNOLOGY 

marked  in  intravenous  injection  and  exhibit  decreasing  severity  in  the 
order  named.  Besredka  estimates  that  by  the  use  of  serum,  approxi- 
mately equivalent  reactions  may  be  produced  by  intravenous  injections 
of  0.05  to  o.i  c.c.,  intrathecal  injections  of  0.066  to  0.125  c.c.  and  intra- 
peritoneal  injections  of  5.0  to  6.0  c.c.  Subcutaneous  injections  in  experi- 
mental animals  rarely  produce  severe  or  fatal  reactions. 

When  used  for  the  intoxicating  dose  the  proteins  are  subject  to  the 
same  physical  and  chemical  agents  as  have  been  discussed  in  connec- 
tion with  the  sensitizing  injection.  The  statement  of  Gay  and  Adler 
that  the  sensitizing  agent  is  contained  in  the  globulin  fraction  of  serum 
and  the  intoxicating  agent  in  the  whole  serum  and  albumin  fractions 
is  not  generally  accepted  and  has  recently  been  contradicted  by  Kato. 
Kato  found  that  guinea-pigs  sensitized  to  any  of  the  serum  fractions 
respond  to  intoxicating  doses  of  any  of  the  fractions  but  most  strongly 
to  that  fraction  to  which  they  were  sensitized.  The  aging  of  serum 
has  an  important  influence.  The  toxicity  of  fresh  serum  decreases 
rapidly  during  the  first  ten  days  of  preservation  to  about  half  its 
original  power.  A  slight  decrease  occurs  during  the  first  two 
months,  after  which  the  deterioration  is  extremely  gradual.  Besredka 
has  found  that  a  serum  twenty  years  old  produced  anaphylactic  shock 
in  a  sensitized  animal.  Uhlenhuth  states  that  he  has  produced  ana- 
phylactic shock  with  proteins  from  mummies. 

The  selection  of  the  route  of  intoxicating  injection  depends  on  the 
character  of  the  protein;  the  intravenous  route  is  undesirable  with 
solid  proteins  and  even  with  bacteria  because  thrombosis  and  embolism 
confuse  the  picture  of  anaphylaxis.  The  minimal  intoxicating  dose  is 
larger  than  the  minimal  sensitizing  dose  in  the  ratio  of  about  100  to  i. 
Wells  has  obtained  fatal  reactions  with  0.000,001  gram  crystallized 
egg-white.  Fatal  reactions  are  rarely  obtained  with  less  than  0.025  c.c. 
serum  and,  as  a  rule,  0.05  c.c.  to  o.i  c.c.  is  required.  We  find  that  for 
laboratory  demonstrations  the  use  of  0.05  c.c.  serum  given  subcu- 
taneously  for  sensitization  and  o.i  c.c.  fresh  serum  given  intravenously 
practically  always  produces  fatal  reactions.  Of  great  importance  is  the 
fact  that  there  is  considerable  individual  variation  not  only  in  the 
sensitivity  of  the  experimental  animals  but  also  in  the  sera  employed 
for  experiments.  Wells  has  stated  that  blood  serum  contains  so  many 
substances  that  it  is  in  reality  an  "  extract  of  the  animal  " ;  hence 
variations  such  as  are  found  in  serum  are  not  present  in  pure  iso- 
lated proteins. 

The  Reaction. — The  phenomena  of  the  reaction  may  be  discussed 
under  three  heads,  the  objective  manifestations,  the  morbid  anatomical 
changes  and  the  functional  disturbances.  The  reaction  may  be  imme- 
diate or  delayed,  depending  upon  the  sensitiveness  of  the  animal,  the 
size  and  mode  of  administration  of  the  toxic  dose  and  the  state  of 
deterioration  of  the  intoxicating  substance.  The  immediate  reaction 
is  called  anaphylactic  shock.  In  the  guinea-pig  the  objective  manifesta- 
tions include  rubbing  of  the  nose,  ruffling  of  the  fur,  evacuation  of 
urine  and  feces,  spasmodic  movements  of  increasing  severity,  including 


PLATE  II. 


Drawing  of  the  gross  appearance  of  the  lungs  of  the 
guinea-pig  in  anaphylactic  shock,  showing  marked 
distention  and  pallor.  Note  the  overlapping  of  the 
lobes  and  the  almost  complete  masking  of  the  heart. 


HYPERSUSCEPTIBILITY  213 

violent  general  convulsions,  marked  inspiratory  and  expiratory  effort 
with  cyanosis,  exhaustion  and  death  from  respiratory  failure  with  the 
heart  still  beating.  In  the  dog  the  respiratory  and  convulsive  phe- 
nomena are  not  so  marked ;  there  is  violent  precordial  activity,  marked 
fall  in  blood-pressure,  diarrhea  and  vomiting.  In  man  the  phenomenon 
may  show  predominance  of  the  respiratory  and  convulsive  symptoms 
or  of  the  cardio-vascular  and  the  gastro-intestinal  symptoms.  Other 
animals  show  variations  of  the  general  picture  outlined.  The  necropsy 
on  a  guinea-pig  shows  large,  pale,  distended  lungs  filling  the  thoracic 
cavity,  cardiac  dilatation,  particularly  of  the  right  side,  passive  con- 
gestion of  the  abdominal  viscera  sometimes  associated  with  minute 
hemorrhages  in  the  gastro-intestinal  tract.  The  lungs  may  show  con- 
gestion, edema  and  small  hemorrhages,  but,  as  a  rule,  the  distention 
is  so  marked  that  there  is  little  blood  in  these  organs.  Microscopically 
there  is  marked  distention  of  the  alveoli,  with  rupture  of  their  walls, 
constriction  of  the  bronchioles  and  frequently  of  the  small  arteries. 
Gay  and  Southard  describe  fatty  degenerative  changes  in  capillary 
endothelium  near  small  hemorrhages,  as  well  as  fatty  changes  in  heart 
muscle,  skeletal  muscle  and  peripheral  nerves.  Beneke  and  Stein- 
schneider  found  Zenker's  degeneration  particularly  of  the  respiratory 
muscles,  but  Wells  believes  this  to  be  the  result  of  asphyxia  which  pro- 
duces Zenker's  hyalin  through  the  increase  of  lactic  acid  in  the  muscle. 
In  dogs  and  other  animals  the  pulmonary  distention  is  not  marked ;  the 
important  features  are  dilation  of  the  heart,  marked  congestion  and 
multiple  hemorrhages.  None  of  these  anatomical  changes  is  charac- 
teristic or  to  be  distinguished  from  other  toxic  conditions.  The  pul- 
monary distention  is  more  distinctive  than  any  of  the  other  changes. 

From  the  functional  point  of  view  there  have  been  extensive  in- 
vestigations of  the  distention  of  the  lungs,  the  fall  of  blood-pressure, 
fall  in  temperature,  delayed  coagulability  of  the  blood  and  alterations 
of  the  nitrogen  metabolism.  Auer  and  Lewis  found  that  in  guinea-pigs 
death  is  due  to  asphyxia  "  apparently  produced  by  tetanic  contraction 
of  the  smooth  muscles  of  the  bronchioles."  This  is  independent  of 
pithing,  section  or  degeneration  of  the  vagus,  and  is  therefore  periph- 
eral, either  in  the  nerve  terminals  or  the  muscle  itself.  Auer  has 
shown  that  atropin  reduces  this  effect,  thus  indicating  the  action  upon 
nerve  terminals.  Karsner  and  Nutt  found  that  there  is  a  definite 
quantitative  relation  between  the  intoxicating  dose  of  serum  and  the 
protective  dose  of  atropin  and  this  fact,  together  with  the  protective 
action  of  anesthetics  such  as  ether,  indicates  that  there  may  be  factors 
involved  other  than  mere  physiological  antagonisms.  The  exciting 
action  on  smooth  muscle  is  not  confined  to  the  bronchiolar  muscle  for 
Schultz  demonstrated  a  similar  action  in  vitro  on  smooth  muscle  of  the 
intestine  and  bladder  of  sensitized  guinea-pigs  and  this  has  subsequently 
been  extended  to  include  other  smooth  muscle  such  as  uterus.  Pelz 
and  Jackson  have  recently  observed  broncho-constriction  in  dogs  during 
the  acute  shock,  but  although  this  is  severe  we  have  been  unable  to 
demonstrate  acute  emphysema  in  dogs. 


214  THE  PRINCIPLES  OF  IMMUNOLOGY 

The  fall  in  blood-pressure  appears  in  anaphylactic  shock  in  the  dog 
and  cat  but  is  not  so  highly  characteristic  of  the  reaction  in  the  guinea- 
pig  or  rabbit.  Biedl  and  Kraus  described  the  condition,  and  it  has 
since  been  studied  extensively.  Pearce  and  Eisenbrey  note  that  it 
amounts  to  a  fall  of  from  20  to  30  mm.  mercury  in  the  dog  and  believe 
it  to  be  due  to  vaso-dilatation,  particularly  of  the  splanchnic  area,  due 
to  action  upon  the  nerve  endings  rather  than  upon  the  muscle.  Schultz 
was  of  the  opinion  that  the  fall  in  pressure  is  due  to  direct  action  upon 
the  heart  by  the  toxic  agent.  He  expressed  the  opinion  that  in  the  cat 
the  fall  in  general  pressure  is  due  to  vaso-constriction  in  the  pulmonary 
circuit  so  that  the  right  heart  cannot  empty  itself.  Eisenbrey  and 
Pearce  in  a  further  study  on  dogs  found  that  the  functional  activity 
of  the  myocardium  is  not  primarily  affected,  that  there  is  no  satisfactory 
evidence  of  pulmonary  vaso-constriction  and  that  the  later  changes 
in  the  myocardium  with  the  fall  in  general  pressure  result  from  incom- 
plete filling  of  the  heart  consequent  on  the  accumulation  of  blood  in 
the  larger  venous  trunks,  particularly  of  the  splanchnic  area.  Simonds 
finds  that  with  the  fall  in  arterial  pressure,  there  is  a  fall  in  pressure  in 
the  superior  vena  cava  and  a  rise  in  portal  vein  pressure,  associated 
with  an  increase  in  the  volume  of  the  liver.  Upon  examination  of  the 
hepatic  vein  of  the  dog  the  vessel  shows  a  very  heavy  musculature  as 
compared  with  that  of  the  herbivorous  animals,  and  Simonds  con- 
cludes that  spasm  of  the  hepatic  vein  and  its  tributaries  explains  the 
phenomena  observed.  Manwaring  and  subsequently  Voegtlin  and 
Bernheim  had  previously  found  that  exclusion  of  the  liver  from  the 
circulation  prevented  the  appearance  of  anaphylactic  shock,  observations 
well  in  accord  with  Simonds'  hypothesis.  However,  Pelz  and  Jackson 
excluded  the  entire  abdominal  circulation,  and  in  spite  of  this  demon- 
strated broncho-constriction  and  marked  fall  in  blood-pressure.  Thus, 
although  numerous  factors  may  play  a  part,  the  only  fact  that  we  can 
bring  forward  as  generally  accepted  is  that  the  fall  in  arterial  pressure 
is  associated  with  peripheral  vaso-dilatation.  Davis  and  Petersen 
observed  an  increase  in  the  volume  of  lymph  for  a  short  time  imme- 
diately following  injection  and  again  for  a  longer  period  beginning 
about  one  hour  after  injection.  The  antiferment  increases  in  the  lymph 
without  any  change  in  the  blood  serum. 

There  can  be  no  doubt  that  the  gaseous  interchange  in  the 
convulsive  phases  of  anaphylactic  shock  is  increased.  Varying  reports, 
however,  have  appeared  as  to  the  influence  of  anaphylactic  shock  upon 
nitrogen  metabolism.  Major  found  an  inconstant  decrease  of  nitrogen 
output  in  rabbits  during  shock,  but  this  increased  in  the  animals  that 
survived  the  immediate  shock  to  such  a  degree  as  to  exceed  the  intake. 
Zunz  and  Gyorgy  found  a  definite  increase  in  amino-acids,  which 
Jobling,  Petersen  and  Eggstein  confirmed,  with  the  additional  informa- 
tion that  the  total  non-coagulable  nitrogen  is  increased.  Hisanobu 
found  a  marked  increase  of  urea  nitrogen,  as  well  as  of  the  non-urea 
and  amino-acid  nitrogen.  He  concludes,  as  would  also  be  apparent 
from  Major's  work,  that  there  is  an  abnormally  rapid  destruction  of 


FIG.   17. — Drawing  of  the  microscopical  appearance  of  the  lung  of  the  guinea-pig 
in  anaphylactic  shock,  showing  the  alveolar  emphysema,  constriction  of  the  bron- 
chioles and  of  an  arteriole. 


HYPERSUSCEPTIBILITY  215 

tissue  proteins.  Jobling,  Petersen  and  Eggstein  found  an  increase  in 
non-specific  protease  with  a  decrease  of  antiferment  and  an  associated 
decrease  of  serum  proteoses ;  this  is  followed  by  a  progressive  increase 
in  non-coagulable  nitrogen,  proteoses  and  serum  lipase.  They,  there- 
fore, conclude  that  "  the  acute  intoxication  is  brought  about  by  the 
cleavage  of  serum  proteins  (and  proteoses)  through  the  peptone  stage 
by  a  non-specific  protease."  Modern  opinion  thus  favors  an  increase 
in  nitrogenous  metabolism  in  anaphylactic  shock  and  this  may  well 
be  due  to  a  liberation  or  mobilization  of  proteases ;  that  the  action  of 
the  latter  is  limited  to  the  blood  appears  to  us  not  to  be  conclusively 
proven.  In  spite  of  the  increase  in  metabolism  there  is  a  fall  in  body 
temperature ;  therefore,  there  must  be  an  increase  in  heat  radiation. 
In  lower  animals  the  respiratory  function  is  of  great  importance  in  heat 
radiation,  and  we  suggest  that  the  marked  increase  of  respiration  in 
anaphylactic  shock  has  some  bearing  on  this  problem,  but  we  by 
no  means  wish  to  exclude  other  factors  that  may  play  a  part  in 
the  phenomenon. 

The  decrease  in  coagulability  of  the  blood  was  first  observed  by 
Biedl  and  Kraus  and  since  has  been  amply  confirmed.  They  believed 
the  change  to  be  due  to  either  a  decrease  of  thromboplastin  or  an 
increase  in  antithrombin.  The  salts  of  the  blood  apparently  are  un- 
changed. Achard  and  Aynaud,  as  well  as  Lee  and  Vincent,  found 
a  decrease  in  the  number  of  platelets,  but  this  was  not  found  by  Biedl 
and  Kraus.  Shattuck  found  a  delay  in  action  of  prothrombin.  Pepper 
and  Krumbhaar  reached  the  same  conclusion  as  Biedl  and  Kraus  con- 
cerning a  decrease  of  thromboplastin  or  an  increase  of  antithrombin. 
Bulger  concludes,  in  terms  which  summarize  our  knowledge  at  the 
present  time,  that  the  decrease  in  coagulability  is  "  due  to  changes  in 
that  stage  of  the  coagulation  process  at  which  thrombin  is  formed 
through  the  interaction  of  prothrombin,  calcium,  thromboplastin 
and  antithrombin  (  ?) .  These  changes  are  probably  due  to  variations 
in  thromboplastin." 

Desensitization  or  Anti-anaphylaxis. — If  an  animal  recovers  from 
anaphylactic  shock  its  earlier  hypersusceptibility  is  replaced  by  a  period 
of  resistance  during  which  injections  of  the  specific  protein  produce  no 
demonstrable  reaction.  This  refractory  period  lasts  for  a  varying 
period  of  time  up  to  several  weeks,  and  although  the  animal  subse- 
quently becomes  hypersusceptible,  it  rarely  reaches  the  same  degree  of 
hypersusceptibility  which  it  primarily  exhibited.  These  facts  were 
pointed  out  in  the  original  investigations  of  Rosenau  and  Anderson 
and  of  Otto.  Besredka  has  studied  the  matter  extensively  and  has 
found  that  very  small  doses  of  the  protein  may  desensitize,  doses  in 
themselves  too  small  to  lead  to  any  observable  symptoms.  By  repeated 
injections  it  is  possible  to  produce  such  a  degree  of  resistance  that  the 
animal  may  withstand  doses  1000  times  as  great  as  that  which  proves 
fatal  if  desensitization  has  not  occurred.  The  rapidity  with  which 
desensitization  appears  depends  upon  the  route  of  injection.  After 
subcutaneous  injection  it  may  not  appear  for  twenty-four  hours ;  intra- 


216  THE  PRINCIPLES  OF  IMMUNOLOGY 

peritoneal  injections  may  require  three  or  four  hours  for  results, 
whereas  intravenous  injections  may  be  effective  in  a  few  minutes.  An 
experiment  quoted  from  Besredka  illustrates  the  rapidity  and  extent 
of  the  process.  Guinea-pigs  sensitized  with  egg-white  exhibited  fatal 
reactions  with  a  toxic  dose  of  0.002  c.c.  egg-white.  An  animal  of  this 
group  was  given  0.0005  c-c-  egg-white  intravenously  without  reaction. 
It  did  not  react  to  0.005  c-c->  tne  ^ata^  dose,  given  two  minutes  after 
the  first  injection  nor  to  doses  of  0.02'  c.c.,  or  0.2  c.c.,  given  at  ten- 
minute  intervals.  Ten  minutes  later  it  was  given  2.0  c.c.  and,  although 
visibly  uncomfortable  for  a  time,  recovered.  Desensitization  may  also 
be  practised  by  four  or  five  repeated  subcutaneous  or  intraperitoneal 
injections  at  intervals  of  about  two  hours,  the  subcutaneous  route 
requiring  a  longer  time  to  be  effective  than  the  intraperitoneal  route. 
We  have  found  relatively  large,  but  still  sub-lethal,  single  doses  to  be 
most  effective  by  intravenous  injections,  but  less  so  by  intraperitoneal 
and  least  by  subcutaneous  injection.  Besredka  also  reports  desensitiza- 
tion  by  introducing  the  protein  into  the  gastro-intestinal  tract  but  as 
yet  this  has  not  received  widespread  confirmation.  Desensitization  may 
be  effective  at  any  period  during  the  hypersusceptible  state.  If  a  second 
dose  be  given  before  hypersusceptibility  appears,  the  condition  may, 
by  subsequent  injections,  become  one  of  increased  resistance 
or  immunity. 

Other  methods  of  preventing  shock  include  the  use  of  atropin  as 
suggested  by  Auer  and  Lewis,  of  adrenalin,  of  chloral  hydrate,  admin- 
istration of  ether,  alcohol,  atoxyl  and  numerous  other  drugs.  Pelz  and 
Jackson  found  adrenalin  most  satisfactory  in  dogs.  Karsner  and  Nutt 
found  that  atropin  sulphate  is  satisfactory  in  guinea-pigs  provided  the 
toxic  dose  of  serum  is  not  too  large.  The  use  of  adrenalin  in  guinea- 
pigs  is  unsatisfactory  because  of  the  pulmonary  hemorrhage  and 
edema  which  it  produces.  Drugs  which  depress  the  excitability  of 
the  smooth  muscle  of  the  bronchioles,  those  which  depress  nerve  activity 
generally  as  the  anesthetics,  and  those  which  tend  to  maintain  blood 
circulation,  are  pharmacologically  adapted  to  the  prevention  of  ana- 
phylactic  shock.  They  do  not  operate  as  effectively  after  the  toxic  dose 
of  protein  has  been  given,  as  they  do  when  given  in  time  to  produce 
physiologic  effects  before  the  onset  of  shock.  Thomson  found  that 
exposure  to  the  X-ray  inhibits  anaphylactic  shock.  Friedberger  and 
Mita  have  suggested  that  anaphylactic  shock  may  be  inhibited  by  very- 
slow  administration  of  the  protein.  Lewis  has  investigated  this  problem 
experimentally  and  by  the  use  of  the  Woodyat  pump  has  found  that 
"  acute  anaphylactic  shock  can  be  prevented  in  sensitized  experimental 
animals  by  giving  otherwise  fatal  doses  of  diluted  antigen  intravenously 
at  very  slow  rates." 

Passive  Anaphylaxis. — As  was  shown  in  1907  by  the  independent 
investigations  of  Nicolle,  Richet,  Otto  and  Friedemann,  the  serum  of 
a  hypersusceptible  animal,  when  injected  into  a  normal  animal,  will 
render  the  latter  also  hypersusceptible.  This  condition  is  transferred 
in  the  serum  rather  than  in  corpuscles  or  tissue  cells.  Passive  ana- 


FIG.  18. — Tracing  from  the  dog  in  anaphylactic  shock.  From 
above  downward  the  tracings  are:  myocardiograph,  blood- 
pressure,  base  line,  membrane  manometer,  base  line,  signal,  time 
in  seconds.  The  down  strokes  of  the  myocardiograph  tracing 
represent  cardiac  contractions.  The  perpendicular  line  was 
drawn  arbitrarily  through  the  blood-pressure  curve  at  the  point 
where  the  fall  began.  Corresponding  points  in  the  myocardio- 
graph and  Hurthle  manometer  tracings  were  measured  and  are 
indicated  by  the  cross  where  the  recording  levers  were  not  in 
accurate  alignment.  (From  Eisenbrey  and  Pearce.  A  study  of  the 
action  of  the  heart  in  anaphylactic  shock  in  the  dog,  Journal  of 
Pharmacology  and  Experimental  Therapeutics,  4,  21.  1912.) 


H  YPERSUSCEPTIBILIT  Y  2 1 7 

phylaxis  may  be  demonstrated  about  four  hours  after  intravenous 
administration  of  the  serum  from  the  hypersusceptible  animal,  about 
twenty-four  hours  after  intraperitoneal  injection  and  from  twenty-four 
to  forty-eight  hours  after  subcutaneous  administration.  It  remains 
at  its  height  for  about  three  or  four  days,  gradually  disappears  in  a 
few  weeks  and  never  exhibits  the  permanence  of  active  anaphylaxis. 
Further  study  by  numerous  investigators  has  shown  that  passive  ana- 
phylaxis arises  as  the  result  of  injection  of  serum  from  an  animal  in 
the  hypersusceptible  state  or  from  an  animal  in  the  "  incubation " 
period  before  sensitization  can  actually  be  demonstrated;  it  may  also 
follow  the  injection  of  the  serum  of  an  animal  in  the  anti-anaphy lactic 
state  and  may  be  produced  by  the  injection  of  an  immune  precipitating 
serum.  In  the  last-named  instance  an  animal  is  immunized  to  the 
particular  protein  for  which  passive  anaphylaxis  is  to  be  produced. 
Doerr  and  Moldovan  pointed  out  this  fact,  and  it  has  been  repeatedly 
confirmed.  Scott  demonstrated  that  the  intensity  of  the  anaphylactic 
shock  parallels  the  titer  of  the  precipitating  serum.  The  young  of 
sensitized  female  guinea-pigs  are  sensitive,  as  has  long  been  known. 
The  recent  work  of  Reinals  confirms  this  fact,  but  does  not  definitely 
settle  the  question  as  to  whether  the  sensitization  of  the  young  is  active 
or  passive. 

Specificity  of  Anaphylaxis. — That  the  process  is  specific  was 
pointed  out  by  the  earliest  investigators.  It  is  undoubtedly  one  of  the 
most  specific  of  the  biological  reactions,  as  is  emphasized  by  its  extreme 
delicacy  in  regard  to  sensitizing  dose.  Nevertheless,  group  reactions 
appear  as  in  the  reactions  of  immunity.  For  example,  a  guinea-pig 
sensitive  to  sheep  serum  will  react  somewhat  less  violently  to  goat 
serum.  Wells  and  Osborne  have  shown  that  cross  reactions  occur 
between  gliadin  from  wheat  and  rye,  and  hordein  from  barley.  The 
reactions,  however,  are  strongest  with  the  homologous  protein.  Never- 
theless, Wells  was  able  to  separate  ovovitellin  and  crystallized  egg-white 
by  the  anaphylaxis  reaction  and  is  of  the  opinion  that  where  group 
reactions  occur  the  reactions  are  the  result  of  common  groups  in  the 
protein  molecules  even  though  the  proteins  may  appear  to  be  chemi- 
cally distinct.  If  guinea-pigs  are  sensitized  to  several  proteins  simul- 
taneously they  will  react  to  any  of  the  proteins  employed,  but  after  an 
animal  has  reacted  to  one  of  the  proteins,  subsequent  reactions  to  the 
others  are  less  severe.  Investigations  conducted  in  this  laboratory  with 
serum  proteins  indicate  that  although  desensitization  is  best  produced 
by  homologous  sera  it  may  be  effected  by  biologically-related  sera  and 
by  non-related  sera.  For  such  purposes  considerably  larger  doses  of  the 
heterologous  sera  are  necessary  than  of  the  homologous  serum.  Against 
the  assumption  that  desensitization  indicates  the  specificity  of  the  reac- 
tion is  the  fact  claimed  by  Banzhaf  and  Steinhardt  that  lecithin  protects 
against  anaphylactic  shock.  Rosenau  and  Anderson  failed  to  confirm  the 
work  of  Banzhaf  and  Steinhardt,  but  it  may  well  be  that  a  colloidal  dis- 
turbance of  some  sort  may  prevent  the  appearance  of  shock  and  that 


218  THE  PRINCIPLES  OF  IMMUNOLOGY 

some  similar  disturbance  may  appear  as  the  result  of  injection  of  heter- 
ologous  sera. 

In  view  of  the  great  specificity  of  anaphylaxis  it  was  hoped  that 
by  this  means  organ  specificity  might  be  demonstrated.  Ranzi  used 
extracts  of  liver,  kidney,  spleen  and  ovary  and  found  that  these  gave 
species  reactions  with  serum,  but  that  there  was  no  evidence  of  organ 
specificity.  Pfeiffer  pointed  out  that  the  organs  employed  by  Ranzi 
contained  blood  and  therefore  could  not  be  expected  to  show  more 
than  species  reactions.  He  washed  the  organs  apparently  free  from 
blood  and  found  that  animals  sensitized  to  a  given  organ  extract  respond 
somewhat  more  markedly  to  that  extract  than  to  extracts  of  other 
organs,  i.e.,  there  is  a  relative  specificity.  This  was  found  to  be  true 
in  somewhat  lesser  degree  by  Pearce,  Karsner  and  Eisenbrey.  Minet 
and  Bruyant  desensitized  with  -serum  and  then  attempted  to  produce 
shock  by  the  organ  extracts ;  they  failed  to  demonstrate  organ  specificity. 
Bell  has  pointed  out  the  fact  that  the  most  careful  perfusion  of  organs 
fails  to  remove  the  blood  completely,  and  it  appears  that  Minet  and 
Bruyant's  conclusions  must  hold  for  the  present.  Extracts  of  sperma- 
tozoa and  of  ovary  fail  to  exhibit  organ  specificity,  but  crystalline  lens 
behaves  as  it  does  in  the  reactions  of  precipitation  and  cytolysis. 
Numerous  investigators  have  shown  that  the  lenses  of  different  species 
react  with  each  other  but  that  serum  fails  to  interact  as  either  sensi- 
tizer  or  intoxicating  body  with  the  serum  of  the  species  from  which 
the  lens  was  taken. 

There  is  no  doubt  that  anaphylaxis  produced  by  bacterial 
emulsions  or  extracts  is  specific,  but  reports  vary  as  to  the  presence 
of  group  reactions.  Delanoe  holds  that  group  reactions  appear, 
whereas  Kraus  and  his  collaborators  maintain  the  absolute  specificity 
of  bacterial  anaphylaxis. 

Theories  of  the  Reaction  of  Anaphylaxis. — In  order  to  avoid  any 
more  confusion  than  is  necessary  it  seems  well  to  review  these  theories 
in  groups  rather  than  in  historical  sequence.  The  most  important 
difference  of  opinion  is  as  to  whether  or  not  a  poisonous  substance  is 
produced  in  the  reaction.  If  not  it  would  appear  to  be  necessary  to 
suppose  that  some  sort  of  reaction  occurs  in  the  cells  of  the  body  or 
in  the  body  fluids,  perhaps  in  the  nature  of  a  liberation  of  energy  on 
the  part  of  the  cells  or  in  some  form  of  disturbed  colloidal  or  enzymatic 
balance  of  the  fluids.  If  a  toxic  substance  is  formed  it  may  be  pro- 
duced in  the  cells  or  in  the  circulating  fluids.  This  may  be  the  result 
of  partial  destruction  of  the  proteins  of  the  body  or  of  the  introduced 
protein,  or  it  may  appear  as  a  new  body  which  is  formed  by  substances 
produced  by  the  first  injection  coming  in  contact  with  the  antigen  upon 
second  injection.  This  summary  gives  the  essentials  of  the  con- 
troversy, and  a  further  elaboration  follows. 

Anaphylactic  Poisons. — Richet  formulated  the  hypothesis  that  the 
primary  injection  of  protein  produces  a  substance  in  the  body  which 
he  named  toxogenine.  Upon  second  injection  the  antigen  is  supposed  to 
combine  with  the  toxogenine  which  has  been  produced  during  the  period 


HYPERSUSCEPTIBILITY  219 

of  incubation  and  forms  a  toxic  substance  named  apotoxlne.  He  com- 
pares the  reaction  to  the  combination  of  amygdaline  and  emulsine  to 
produce  hydrocyanic  acid.  This  hypothesis  resembles  somewhat  that 
of  Friedberger,  which  has  been  investigated  intensively  by  many 
workers.  Friedberger  prepared  a  toxic  substance,  which  he  named 
anaphylatoxin,  by  mixing  antigen,  precipitating  serum  and  complement. 
He  obtained  a  precipitate  by  mixing  sheep  serum  with  a  specific  immune 
precipitating  serum  from  the  rabbit.  This  precipitate  was  washed,  sus- 
pended in  fresh  guinea-pig  serum  for  twelve  hours,  then  centrifuged. 
The  supernatant  fluid  was  found  to  be  extremely  toxic  for  guinea-pigs. 
The  reduction  of  complement  in  anaphylaxis  has  been  emphasized  by 
Friedberger  in  the  development  of  his  hypothesis  concerning  ana- 
phylatoxin. Thomson,  however,  has  found  that  this  reduction  is  not 
constant  and  that  it  is  in  proportion  to  the  quantity  of  the  free  antibodies 
in  the  circulation.  It  is  insignificant  when  the  animal  has  been  sensi- 
tized with  a  small  single  dose  of  antigen,  but  if  the  animal  has  been 
sensitized  by  repeated  doses  and  the  precipitin  content  of  the  blood  is 
high,  the  diminution  in  complement  is  likely  to  be  marked.  The  symp- 
toms following  injection  of  anaphylatoxin  include  the  usual  clinical 
manifestations,  with  fall  of  temperature,  retardation  of  coagulation  of 
the  blood  and  leucopenia.  The  poison  resists  heat  at  56°  C.  for  one-half 
hour,  resists  desiccation  and  is  precipitated  by  alcohol.  Subsequently 
it  was  found  that  bacteria  and  their  antisera  could  be  employed  in  the 
same  fashion  as  the  precipitinogen  and  precipitin.  Doerr  and  Russ 
found  that  precipitates  are  toxic  without  the  addition  of  complement, 
and  in  view  of  this  fact  and  the  production  of  passive  anaphylaxis  by 
precipitating  sera,  reached  the  conclusion  that  precipitin  and  the  sub- 
stance produced  by  the  primary  injection  in  anaphylaxis  are  inseparable. 
Kraus  and  his  co-workers  have  contradicted  this  parallelism  and  point 
out  that  the  guinea-pig  is  a  poor  producer  of  precipitin;  rabbits  may 
produce  a  powerful  anaphylactic  substance  without  producing  precipi- 
tins;  goats  produce  precipitin  readily  but  have  a  serum  incapable  of 
conferring  passive  anaphylaxis.  Biedl  and  Kraus  pointed  out  the  fact 
that  injections  of  pepton  produce  symptoms  similar  to  anaphylaxis  in 
the  dog.  Karsner  has  confirmed  this  in  the  guinea-pig.  Biedl  and 
Kraus  found  that  following  injection  of  pepton  into  a  dog  the  animal 
subsequently  fails  to  react  to  anaphylaxis  and  hence  they  formulatedV. 
the  hypothesis  that  the  poison  of  anaphylaxis  is  a  pepton-like  body. 
Doerr  offered  the  hypothesis  that  the  actual  disturbance  is  in  the  physi- 
cal character  of  the  blood.  He  assumes,  however,  that  this  disturbance 
is  produced  by  a  toxic  agent  originating  in  complement.  The  com- 
plement is  supposed  to  contain  the  toxic  substance  held  in  check  by  an 
antagonistic  substance ;  the  latter  is  adsorbed  by  precipitates  or  bacteria, 
thus  liberating  the  toxic  substance.  The  further  investigation  of  the 
so-called  anaphylatoxin  led  to  the  discovery  by  Keysser  and  Wasser- 
mann  that  a  similar  substance  could  be  produced  by  the  action  of  com- 
plement on  barium  sulphate  or  kaolin.  Besredka  then  found  that  placing 
fresh  serum  upon  pepton  agar  produces  a  toxic  fluid  which  induces 


220  THE  PRINCIPLES  OF  IMMUNOLOGY 

symptoms  identical  with  those  from  anaphylatoxin.  Bordet  found  that 
the  action  of  fresh  serum  upon  agar  in  solution  produces  a  similar 
toxic  substance.  Novy  and  De  Kruif  have  published  very  extensive 
studies  upon  toxic  materials  in  a  measure  similar  to  the  anaphylatoxin. 
It  is  found  that  the  action  of  serum  upon  agar  intensifies  the  toxic 
power  of  the  agar.  They  have  shown  that  agar  and  other  non-protein 
colloids  produce  anaphylactoid  symptoms. 

The  poison,  if  there  be  such  in  anaphylaxis,  is  not  dependent  on  the 
presence  of  any  antibody  or  other  substance  within  the  cells  of  the  sensi- 
tized animal,  because  it  can  be  produced  in  vitro;  neither  is  it  dependent 
on  antigen,  inasmuch  as  barium  sulphate  and  kaolin  serve  a  similar  pur- 
pose; nor  is  it  dependent  on  complement,  for,  as  Doerr  has  shown,  it 
can  be  produced  without  the  action  of  fresh  serum.  Besredka  maintains 
that  the  anaphylatoxin  produces  no  symptoms  by  sub-dural  injection 
and  that  it  kills  only  upon  intravenous  injection.  Besredka  has  found 
that  pepton  does  not  interfere  with  true  anaphylaxis  in  the  guinea- 
pig,  but  that  it  does  inhibit  the  action  of  anaphylatoxin.  Furthermore, 
the  state  of  anti-anaphylaxis  which  protects  an  animal  against  a  massive 
dose  of  the  antigenic  substance  and  therefore  prevents  anaphylactic 
shock  has  no  such  protective  influence  upon  anaphylatoxin.  These 
arguments  as  well  as  those  presented  in  the  subsequent  section  on  the 
cellular  theories  of  anaphylaxis  serve  to  show  that  there  is  prob- 
ably no  poison,  which  can  be  produced  in  vitro,  that  leads  to  the 
development  of  a  condition  identical  with  true  anaphylaxis.  Certain 
features  of  this  discussion  will  be  referred  to  under  the  heading  of 
Anaphylactoid  Phenomena. 

Cellular  Theories. — The  conflicting  views  are  that  either  a  poison 
is  produced  within  cells,  or  that  some  disturbance  of  cells  appears  inde- 
pendently of  the  production  of  a  poisonous  substance.  Gay  and 
Southard,  influenced  perhaps  by  the  prevailing  conceptions  of  immune 
reactions  and  impressed  by  the  cellular  degenerations  seen  in  their 
animals,  emphasized  the  intracellular  character  of  the  reaction.  They 
assumed  that  the  injected  protein  contains  a  substance,  anaphylactin, 
which  is  eliminated  from  the  body  extremely  slowly,  in  contrast  to  the 
fairly  rapid  elimination  of  the  other  constituents  of  the  protein.  "  The 
anaphylactin,  however,  remains  and  acts  as  a  constant  irritant  to  the 
body  cells,  so  that  their  avidity  for  the  other  assimilable  elements 
of  the  horse  serum  (or  protein),  which  have  accompanied  the  ana- 
phylactin, becomes  enormously  increased.  At  the  end  of  two  weeks  of 
constant  stimulation  on  the  part  of  the  anaphylactin,  and  of  constantly 
increasing  avidity  on  the  part  of  the  somatic  cells,  a  condition  has 
arrived  when  the  cells,  if  suddenly  presented  with  a  large  amount  of 
horse  serum,  are  overwhelmed  in  the  exercise  of  their  increased  assim- 
ilating functions  and  functional  equilibrium  is  so  disturbed  that  local 
or  general  death  may  occur."  This  theory  was  supported  by  their 
statement  that  the  sensitizing  fraction  of  serum  is  contained  in  the 
globulin  fraction  and  that  the  other  elements  of  serum  may  serve  to 
produce  shock.  They  could  not  produce  a  toxic  body  by  mixing  the 


H  YPERSUSCEPTIBILIT  Y  22 1 

serum  of  sensitized  guinea-pigs  and  horse  serum.  The  fact  that 
further  investigation,  as  for  example  that  of  Wells  and  of  Kato,  has 
failed  to  demonstrate  a  manifest  difference  between  sensitizing  and 
intoxicating  fractions  of  the  protein,  is  an  argument  against  this 
hypothesis.  Friedberger's  original  conception  was  that  the  primary 
injection  leads  to  the  development  of  receptors  in  the  cells  but  in  such 
small  amounts  as  not  to  be  liberated  into  the  blood  stream.  These 
"  sessile  "  receptors  are  responsible  for  an  increased  affinity  of  the 
cells  for  the  antigen,  the  consequent  disturbances  resulting  from  the 
rapid  anchoring  of  the  protein  by  the  cells.  If  injections  are  repeated 
before  the  anaphylactic  state  is  developed  the  receptors  are  formed  in 
large  amounts  and  appear  in  the  blood  stream  as  precipitins.  This 
hypothesis  accords  well  with  the  modern  conception  of  immunity  and 
anaphylaxis  save  for  the  assumption  that  the  sensitizing  substance  and 
precipitins  are  identical.  This  theory  was  followed  by  Friedberger's 
anaphylatoxin  theory.  Somewhat  more  concrete  is  the  hypothesis  of 
Vaughan  and  Wheeler.  After  a  long  period  of  study  of  toxic  frac- 
tions of  bacterial  and  other  proteins  by  Vaughan  and  his  co-workers, 
the  following  statement  in  regard  to  anaphylaxis  was  made.  "  When 
a  foreign  protein  is  introduced  into  the  blood  or  tissues  it  stimulates 
certain  body  cells  to  elaborate  the  specific  ferment  which  will  digest 
that  specific  protein.  When  this  protein  first  comes  in  contact  with 
the  body  cells,  the  latter  are  unprepared  to  digest  the  former,  but  this 
function  is  gradually  acquired.  The  protein  contained  in  the  first 
injection  is  slowly  digested,  and  no  ill  effects  are  observable.  When 
subsequent  injections  of  the  same  protein  are  made,  the  cells  prepared 
by  the  first  injection  pour  out  the  specific  ferment  more  promptly,  and 
the  results  are  determined  by  the  rapidity  with  which  digestion  takes 
place.  The  poisonous  group  in  the  molecule  may  be  set  free  rapidly, 
and  in  amounts  sufficient  to  produce  symptoms,  or  to  kill  the  animal." 
Jobling  and  his  co-workers,  however,  have  reached  the  conclusion  that 
the  development  of  proteases  in  the  blood  is  not  dependent  upon  anti- 
bodies and  is  not  specific.  Vaughan  replies  to  this  objection  that  "  we 
have  only  transferred  the  problem  of  specificity  from  the  development 
of  a  specific  enzyme  to  the  specific  uncovering  of  a  non-specific 
enzyme."  Undoubtedly,  the  bodies  studied  by  Vaughan  are  extremely 
toxic.  As  an  example,  he  found  that  the  product  of  I  gram  of  casein 
is  sufficient  to  kill  800  guinea-pigs.  We  are  not  ready  to  admit  that 
toxic  substances  of  this  sort  produce  clinical  and  pathological  changes 
that  are  identical  with  anaphylaxis.  Weil  has  given  the  participation 
of  the  cells  most  extensive  study.  He  considered  that  the  cells  are  of  the 
utmost  importance  in  the  destruction  and  elimination  of  foreign  protein 
and  that  in  the  course  of  this  process  they  construct  an  antibody.  The 
union  of  antigen  and  antibody  within  the  cells  gives  rise  to  the  serious 
disturbances  which  constitute  anaphylaxis.  His  excellent  work  was 
interrupted  by  his  death  in  the  service  of  his  country,  but  his  hypothesis 
is  one  which  serves  equally  well  in  the  phenomenon  of  desensitization 
and  in  anaphylaxis.  In  support  of  the  assumption  that  the  primary 


222  THE  PRINCIPLES  OF  IMMUNOLOGY 

change  is  in  the  cells  may  be  considered  the  work  of  Schultz,  Dale, 
Woods  and  others  with  isolated  sensitized  organs  containing  smooth 
muscle.  These  organs,  washed  free  of  blood,  responded  specifically  to 
the  protein  with  which  the  animal  was  sensitized.  Of  considerable 
value  was  the  experiment  of  Pearce  and  Eisenbrey,  who  transfused 
dogs  so  that  the  blood  of  a  sensitized  dog  circulated  in  the  body  of  a 
normal  dog  and  vice  versa.  Under  these  circumstances  the  intoxicating 
dose  of  the  antigenic  protein  produced  symptoms  in  the  sensitized  dog 
with  normal  blood,  but  no  symptoms  in  the  normal  dog  provided  with 
blood  from  its  sensitized  fellow.  Coca  confirmed  this  with  the  guinea- 
pig.  Although  Manwaring  and  collaborators  have  found  that  per- 
fusion  of  rabbit  heart  indicates  that  anaphylactic  shock  is  entirely 
humoral,  subsequent  work  of  Manwaring  and  Kusama  with  perfusion 
of  guinea-pig  lungs  showed  that  the  cells  of  the*  lungs  of  sensitized 
animals  respond  by  bronchiolar  constriction  to  perf  usion  with  antigenic 
serum.  They  also  found  that  perfusion  of  normal  lungs  with  a  mixture 
of  the  blood  of  a  sensitive  animal  and  antigen  also  produces  bronchiolar 
constriction.  None  of  the  experiments  so  far  outlined  establishes 
definitely  the  parts  the  cells  play,  for,  as  Bell  points  out,  none  of  these 
methods  has  completely  removed  the  native  blood  from  the  organs. 
We  know  that  minute  amounts  of  certain  protein  poisons  are  highly 
toxic,  and  it  may  be  that  the  amount  of  blood  left  in  a  perfused  organ 
is  sufficient  for  the  production  of  a  humoral  poison.  Nevertheless, 
studies  of  passive  anaphylaxis  tend  to  confirm  the  conception  of  cellu- 
lar participation.  Weil  has  pointed  out  that  simultaneous  injection  of 
a  serum,  capable  of  producing  passive  anaphylaxis,  and  its  antigen  fails 
to  produce  symptoms.  A  certain  interval  of  time  must  elapse  before  an 
animal  becomes  passively  anaphylactic,  an  interval  in  which  it  is  pre- 
sumed the  cells  either  anchor  or  develop  the  sensitizing  substance. 
Isolated  organs  fail  to  respond  to  the  antigen  until  a  certain  time  has 
elapsed.  The  time  element  depends  to  a  certain  extent  upon  the  mode 
of  injection,  but  is  never  less  than  several  hours  even  with  intravenous 
injection.  This  fact,  in  association  with  the  experiments  in  active 
anaphylaxis,  in  vitro,  with  perfusion  and  with  isolated  organs,  all  tend 
to  support  the  conception  that  the  participation  of  the  cells  is  of  funda- 
mental importance  in  the  reaction.  Weil  has  studied  further  the  phe- 
nomenon of  desensitization  and  finds  that  the  reaction  between  the 
cellular  antibody  and  the  antigen  follows  in  a  general  wray  the  Danysz 
phenomenon  (see  page  50).  By' the  fractional  injection  of  antigen  the 
substance  in  the  cells  takes  up  the  antigen  so  that  subsequent  additions 
of  antigen  produce  little  effect.  He  states  that  "partially  combined 
cellular  antibody  manifests  a  marked  diminution  in  its  affinity  for  fresh 
antigen."  Thus  the  conception  of  cellular  participation  fits  the  demon- 
strated facts  of  passive  anaphylaxis. 

Physical  Theories. — These  have  been  less  susceptible  to  experi- 
mental proof  than  other  theories  because  of  the  limitations  of  technic. 
As  has  been  mentioned,  Doerr  conceived  the  idea  that  adsorption  of  the 
supposed  antagonistic  substance  of  complement  by  bacteria  or  precipi- 


HYPERSUSCEPTIBILITY  223 

tates  liberates  the  toxic  substance  of  complement.  This  theory  omits 
consideration  of  the  cellular  participation  and  needs  further  elaboration 
to  be  acceptable.  Of  more  significance  is  the  fact  demonstrated  by 
Jobling,  Petersen  and  Eggstein  that  anaphylactic  shock  "  is  accom- 
panied by  (a)  the  instantaneous  mobilization  of  a  large  amount  of 
non-specific  protease,  (b)  a  decrease  of  antiferment,  (c)  an  increase 
in  non-coagulable  nitrogen  of  the  serum,  (d)  an  increase  in  amino- 
acids,  (e)  a  primary  decrease  in  serum  proteoses."  They  conclude  that 
the  "  intoxication  is  brought  about  by  the  cleavage  of  serum  proteins 
(and  proteoses)  through  the  pepton  stage  by  a  non-specific  protease" 
and  that  "  the  specific  elements  lie  in  the  rapid  mobilization  of  this 
ferment  and  the  colloidal  serum  changes  which  bring  about  the  change 
in  antiferment  titer."  From  our  discussion  of  the  cellular  participa- 
tion in  anaphylaxis  the  conception  of  Jobling  cannot  be  accepted  as 
entirely  satisfactory,  but  it  has  more  ground  in  demonstrated  fact  than 
any  of  the  other  physical  theories.  Support  for  Jobling's  conception 
is  furnished  by  Bronfenbrenner  and  others.  Bronfenbrenner  finds, 
however,  that  the  state  of  dispersion  of  the  colloids  is  important  in 
maintaining  the  ferment-antiferment  balance  and  that  simply  bubbling 
ether  through  serum  decreases  the  antitryptic  activity  probably  because 
of  an  increased  dispersion  of  colloidal  particles.  A  similar  decrease 
of  antitryptic  activity  of  the  blood  follows  a  mixture  of  antibodies  and* 
antigen.  This  theory  may  be  applied  to  desensitization  by  assuming 
that  the  small  intoxicating  dose  inhibits  antiferment,  that  the  proteases 
then  operate  and  that  the  split  products  act  as  antitrypsin,  thus  prevent- 
ing the  toxic  effects  of  subsequent  injections.  Danysz  hypothesizes  that 
anaphylaxis  is  an  intracellular  or  intravascular  disturbance  of  digestion 
or  a  combination  of  the  two.  The  disturbance  of  digestion  consists  in  an 
inability  of  the  organism  rapidly  to  transform  the  colloid  antigen  into 
crystalloids.  The  symptoms  are  produced  by  a  sudden  alteration  of 
equilibrium  between  the  sol.  and  gel.  state  of  the  colloids  which  enter 
into  the  composition  of  the  cells  and  of  the  blood.  His  conclusion  that 
acute  anaphylaxis  is  due  to  intravascular  changes  in  the  animal  is  in 
contradiction  to  what  we  believe  to  be  well  demonstrated  facts.  Krits- 
chewsky  found  that  the  sap  of  a  certain  plant,  cotyledon  scheideckeri, 
precipitates  blood  proteins,  agglutinates  and  hemolyzes  erythrocytes. 
Symptoms  in  animals  following  injections  into  the  circulation  or  sub- 
cutaneously  resemble  anaphylaxis  and  are  due,  Kritschewsky  believes,  to 
a  change  in  degree  of  disperseness  of  the  plasma  colloids,  and  he 
therefore  assumes  that  anaphylaxis  is  of  the  same  nature.  We  do  not 
concede  that  Kritschewsky  worked  with  true  anaphylactic  shock,  as 
the  pathological  findings  in  his  animals  lack  the  uniformity  of  those 
seen  in  true  anaphylaxis.  Similarly  we  object  to  the  experiments  of 
Doerr  and  Moldovan  who  produced  toxic  symptoms  by  the  injection 
of  water  colloidal  solutions  of  silicic  acid,  also  of  nucleinic  acid  and 
of  dialyzed  iron  hydroxid.  Kopaczewski  found  that  the  injection  of 
serum  rendered  toxic  by  addition  of  bacterial  suspension  or  colloidal 
gels.,  when  injected  into  animals  reduces  the  surface  tension  of  their 


224  THE  PRINCIPLES  OF  IMMUNOLOGY 

blood  three  or  four  dynes,  from  which  reduction  the  animal  gradually 
recovers.  Upon  investigation  of  the  electrical  potential  of  sera  it  was 
found  that  a  current  of  eight  volts  shows  a  precipitate  at  both  electrodes 
in  the  case  of  normal  serum,  but  that  with  a  so-called  anaphylactic  serum 
the  precipitate  collects  almost  entirely  in  the  negative  pole.  Although 
Besredka's  former  idea  that  the  injected  serum  contains  a  separate 
sensibilisinogen  which  leads  to  the  formation  of  sensibilisin  in  the  cells, 
and  an  antisensibilisin  which  combines  with  sensibilisin  upon  the  second 
injection  of  the  serum  is  not  in  accord  with  prevailing  ideas,  yet  he  was 
one  of  the  first  to  propose  a  physical  theory.  Thus  he  stated  in  a  gen- 
eral way  the  majority  of  the  facts  seem  to  indicate  that  the  phenom- 
ena of  anaphylaxis  and  anti-anaphylaxis  are  reduced  to  the  action  of 
precipitation  and  adsorption  which  upset  the  mutual  relations  of  the 
colloids.  Besredka  no  longer  insists  upon  the  separation  of  the  two 
elements  of  protein,  but  is  of  the  opinion  that  the  important  site  of 
reaction  is  in  the  nerve  cells.  He  believes  that  the  second  injection 
of  the  protein  meets  with  the  preformed  sensibilisin  in  the  cells  and 
produces  there  either  a  liberation  or  absorption  of  energy,  thermal  or 
otherwise,  and  that  this  reaction  leads  to  the  phenomena  O'f  anaphylactic 
shock.  He  compares  the  reaction  to  the  mixing  of  water  and  sulphuric 
acid.  If  the  water  is  added  suddenly  to  the  acid  there  is  an  explosive 
liberation  of  the  heat  of  hydration.  If  the  water  is  added  slowly,  the 
heat  is  generated  more  gradually  and  no  serious  manifestations  take 
place.  So  with  anaphylaxis,  if  the  injection  is  in  a  single  large  dose, 
anaphylactic  shock  is  produced,  but  if  several  small  doses  are  given, 
there  is  a  series  of  very  slight  shocks  leading  to  no  serious  disturbance 
and  so  desensitizing  the  body  that  serious  results  cannot  follow  a 
subsequent  large  injection.  Besredka  argues  that  the  inhibitory  effect 
of  anesthetics  supports  his  contention  that  the  nerve  cells  are  of  great 
importance  in  production  of  anaphylactic  shock,  but  the  work  of  numer- 
ous investigators  shows  that  the  broncho-constriction  and  fall  in  blood- 
pressure  occur  in  spite  of  anesthesia,  and  that  the  reaction  may  be  fatal 
if  the  intoxicating  dose  be  sufficiently  large.  Bronfenbrenner  points 
out  that  anesthetics  increase  the  antitryptic  power  of  the  blood  100 
per  cent,  or  more,  thus  inhibiting  the  liberation  of  proteases  and  the 
consequent  production  of  toxic  split  products.  As  a  further  objection 
to  Besredka's  conception  is  the  fact  that  the  experiments  with  isolated 
organs  and  perfusion  demonstrate  that  smooth  muscle  reacts  and  that 
the  phenomenon  is  by  no  means  confined  to  the  nerve  cells.  When 
calorimetric  and  metabolism  experiments  can  be  performed  with  nerve 
tissues,  definite  information  can  be  obtained  in  regard  to  energy  changes 
in  these  tissues  in  anaphylaxis. 

Anaphylactoid  Phenomena. — In  the  discussion  of  the  theories  of 
anaphylaxis  references  have  been  made  to  anaphylatoxin  and  certain 
similar  substances.  As  has  been  pointed  out,  these  substances  may  be 
protein  in  character,  may  represent  certain  decomposition  products  of 
protein,  or  may  be  non-protein  colloids.  It  is  even  maintained  that 
arsphenamine  is  to  be  included  in  this  category  of  colloids.  The  in- 


HYPERSUSCEPTIBILITY  225 

vestigation  of  these  substances  has  had  an  important  bearing  on  the 
development  of  theories  of  anaphylaxis,  because  if  these  can  be  com- 
pared to  the  supposed  toxic  substance  of  anaphylaxis  it  would  seem 
reasonable  to  suppose  that  anaphylaxis  has  as  its  basis  a  colloidal  dis- 
turbance. Many  of  those  who  have  worked  with  these  substances 
have  not  been  strict  in  their  use  of  the  term  anaphylaxis  and  have 
depended  in  large  part  on  the  clinical  manifestations  following  the  injec- 
tions of  these  agents.  From  time  to  time  certain  investigators  have 
indicated  that  more  intimate  study  would  prove  that  anaphylaxis  and 
the  phenomena  following  the  injection  of  these  colloids  are  not  identi- 
cal. Manwaring  and  Crowe,  for  example,  found  that  occasionally  there 
appears  in  anaphylaxis  occlusion  of  pulmonary  blood-vessels  by  thrombi 
and  used  the  term  pseudo-anaphylaxis.  The  problem  has  recently  been 
investigated  extensively  by  Hanzlik  and  Karsner.  The  experiments 
in  this  series  oi  studies  were  controlled  by  gross  and  microscopic  studies 
of  the  viscera  of  the  animals  after  death.  More  than  thirty  colloidal 
agents  were  studied  by  a  variety  of  methods,  including  intravenous 
injection,  studies  of  perfused  organs,  protection  by  atropin  and  epi- 
nephrin,  as  well  as  test-tube  studies  of  the  action  of  the  agents  upon 
blood-corpuscles.  Many  of  the  agents  studied  produce  serious  dis- 
turbances of  circulation  and  others  produce  equally  serious  disturb- 
ances of  respiration.  In  the  case  of  none  of  these  colloids  was  it 
possible  to  demonstrate  that  the  clinical  and  morbid  anatomical  phe- 
nomena, taken  collectively,  are  identical  with  those  of  anaphylaxis. 
The  symptoms  provoked  can  all  be  explained  on  grounds  other  than 
the  assumption  that  we  are  dealing  with  anaphylaxis.  Even  in  the  case 
of  agar,  where  bronchial  constriction  and  pulmonary  distention  are  well 
marked,  the  common  occurrence  of  thrombi  both  in  the  living  animal 
and  in  perfused  lungs  definitely  excludes  an  identity  with  anaphylaxis. 
These  phenomena  may,  therefore,  be  considered  as  of  colloidal  nature 
and  may  well  be  referred  to  as  "  colloid  shock."  Pepton  produces  symp- 
toms and  signs  more  nearly  like  those  of  true  anaphylaxis  than  the  other 
substances  studied,  but  the  fact  that  pepton  more  frequently  produces 
thrombosis,  hemorrhage  and  edema  of  the  lungs  than  is  the  case  in  true 
anaphylaxis,  would  place  pepton  poisoning  in  the  group  of  ana- 
phylactoid  rather  than  anaphylactic  phenomena.  Similarly  the  injec- 
tion of  primarily  toxic  sera  such  as  ox  serum  and  eel  serum  into  the 
guinea-pig  produces  certain  circulatory  disturbance  with  hemorrhage 
and  edema.  It  seems  probable  that  the  toxicity  of  some  of  the  sub- 
stances of  protein  nature  or  the  decomposition  products  of  protein  may 
depend  for  their  activity  upon  the  presence  of  histamine.  The  poison- 
ous character  of  histamine  depends  in  no  way  upon  previous  sensitiza- 
tion,  is  primarily  toxic  and  therefore  may  be  included  as  anaphylactoid 
in  its  action. 

Summary. — With  a  strict  adherence  to  the  conception  that  ana- 
phylaxis constitutes  that  state  of  hypersusceptibilityto  a  given  substance, 
which  has  been  induced  by  a  previous  injection  of  the  same  substance 
we  may  conclude  that  the  mode  of  the  second  injection  determines  the 
15 


226  THE  PRINCIPLES  OF  IMMUNOLOGY 

particular  manifestations  observed.  The  sensitizing  fraction  of  the 
protein,  if  there  be  any  such  fraction,  has  not  been  isolated  nor  has 
the  intoxicating  substance.  If  the  second  dose  be  given  in  mass,  ana- 
phylactic  shock  results.  If,  on  the  other  hand,  divided  small  doses  are 
given  the  state  of  the  organism  is  so  changed  that  severe  anaphylactic 
shock  does  not  appear.  In  agreement  with  Besredka,  we  believe  that 
desensitization  produces  a  series  of  minor  shocks  but  believe  that  the 
explanation  lies  rather  in  the  work  of  Weil  than  in  the  hypothesis  of 
Besredka.  In  other  words,  there  is  a  partial  saturation  of  the  sensi- 
tizing substance  within  the  cells,  so  that  any  subsequent  union  cannot 
produce  the  intensity  of  reaction  that  would  have  been  produced  by  a 
massive  injection.  The  time  that  must  elapse  for  the  production  of 
passive  anaphylaxis,  as  well  as  the  other  experiments  offered  in  evi- 
dence, support  the  conception  that  some  change  must  occur  in  the  cells 
in  order  to  produce  sensitization.  The  nature  of  the  combination 
between  the  specific  protein  and  the  substance  within  the  cells  or  the 
influence  of  the  protein  upon  the  cells  is  not  definitely  known,  but  the 
data  offered  in  review  appear  to  rule  out  the  probability  that  definite 
toxic  bodies  are  formed.  Similarly  the  nature  of  the  primary  changes 
in  the  cells  upon  second  injection  cannot  be  identified;  as  to  whether 
there  is  a  liberation  of  energy  of  some  sort  or  a  disturbance  of  colloidal 
relations  must  still  be  the  subject  of  investigation.  The  specificity  of 
the  reaction  is  similar  to  that  of  other  biological  reactions  and  is  subject 
to  similar  limitations  of  the  group  phenomenon.  Nevertheless,  we  find 
in  anaphylaxis  a  most  specific  phenomenon,  which  is  approached 
in  delicacy  only  by  the  reactions  of  precipitation  and  of  com- 
plement fixation. 

The  Relation  of  Anaphylaxis  to  Immunity. — If  desensitization  of 
an  anaphylactic  animal  is  carried  on  for  only  a  short  time  the  period  of 
desensitization  is  relatively  brief,  but,  on  the  other  hand,  if  the  vac- 
cination be  continued  the  animal  may  be  rendered  resistant.  This  indi- 
cates a  close  relationship  between  the  two  phenomena.  We  do  not 
propose  to  discuss  this  at  length  because  of  the  intricacy  of  the  subject. 
Weil  pointed  out  in  his  earlier  experiments  by  saturation  of  the  animal 
with  proteins  that  although  the  animal  may  become  immune  in  so  far 
as  his  body  fluids  are  concerned  he  may  remain  hypersensitized  in  so 
far  as  his  cells  are  concerned.  Manwaring  and  Kusama  found  that 
the  lungs  of  guinea-pigs  immunized  to  a  certain  protein,  when  washed 
free  of  blood,  were  still  sensitive  to  perfusion  with  the  protein  in 
question.  We,  therefore,  revert  to  the  conception  of  Weil  that  im- 
munity is  in  large  part  exhibited  in  protective  power  of  the  blood  and 
body  fluids.  In  the  state  of  anaphylaxis  this  immunity  has  not  been 
established  in  the  fluids  and  therefore  the  cells  can  be  directly  operated 
upon  by  the  antigen.  If,  on  the  other  hand,  the  animal  is  immune  his 
blood  and  fluids  combine  with  the  antigen  so  as  to  protect  the  cells. 
The  direct  bearing  of  this  upon  diseases  in  man  is  a  matter  of  specula- 
tion. It  seems  possible,  however,  that  during  the  period  of  incubation 
of  an  infectious  disease  the  animal,  as  suggested  by  Danysz,  likewise 


HYPERSUSCEPTIBILITY  227 

passes  through  the  period  of  sensitization  to  the  infecting  organism. 
When,  therefore,  the  infecting  organism  or  its  products  are  present  in 
sufficient  amounts  the  manifestations  of  disease  appear  in  the  form  of 
what  may  be  termed  an  acute  or  sub-acute  anaphylaxis.  As  time  goes 
on  this  process  is  transformed  into  an  immunity  and  the  disease  under- 
goes cure.  In  this  latter  state  it  may  be  assumed  that  the  body  fluids 
have  developed  a  sufficient  amount  of  protective  substance  so  that  the 
cells  are  no  longer  susceptible  to  attack.  This  would  satisfactorily 
explain  the  self -limitation  of  infectious  disease.  By  assuming  that 
injury  of  the  cells  may  have  become  so  serious  as  completely  to 
interfere  with  life  processes,  death  may  ensue,  or  if  the  disturb- 
ance is  not  so  severe  the  condition  may  exhibit  the  chronic  com- 
plications which  so  frequently  follow  acute  infection. 


CHAPTER  XI 
HYPERSUSCEPTIBILITY  IN  MAN 

INTRODUCTION. 

SERUM   DISEASE. 

THE  DELAYED  REACTION. 
THE  ACCELERATED   REACTION. 
ANAPHYLACTIC   SHOCK  IN   MAN, 
NATURAL  HYPERSUSCEPTIBILITY. 

TESTS  FOR  HYPERSUSCEPTIBILITY. 

TOXINS  IN  HAY  FEVER. 
TECHNIC  OF  CUTANEOUS  TESTS. 
DELICACY  OF  TESTS. 
THE  REACTION. 

THEORIES  OF   CUTANEOUS   REACTION.  ^ 

DRUG  IDIOSYNCRASIES. 
THE  TUBERCULIN  TEST. 

GENERAL  REACTION. 
CUTANEOUS   REACTION. 
INTRACUTANEOUS  TEST. 
CONJUNCTIVAL  TEST. 
THEORIES  OF  TUBERCULIN  TEST. 
SPECIFICITY. 
UTILITY. 

THE  LUETIN  REACTION. 

CUTANEpUS   REACTIONS   IN   TYPHOID  FEVER. 
CUTANEOUS   REACTIONS   IN   GONOCOCCUS  INFECTIONS. 
CUTANEOUS   REACTIONS   IN    MENINGOCOCCUS    INFECTIONS. 
CUTANEOUS   REACTIONS   IN    PNEUMOCOCCUS    INFECTIONS. 
CUTANEOUS   REACTIONS   TO  VACCINE   VIRUS. 
CUTANEOUS   REACTIONS    IN   GLANDERS. 
OTHER  CUTANEOUS  REACTIONS. 

Introduction. — The  manifestations  of  hypersusceptibility  in  man 
can  be  classified  into  two  groups,  those  in  which  a  definite  previous  sensi- 
tization  has  been  effected  and  those  in  which  no  such  sensitization  is 
known  or  can  be  conclusively  proven.  In  the  former  group  are 
included  a  relatively  few  cases  of  anaphylactic  shock  and  the  widely- 
observed  phenomenon  called  serum  disease.  In  the  latter  group  are 
those  individuals  who  are  abnormally  sensitive  to  a  wide  variety  of  sub- 
stances. These  may  gain  access  to  the  body  from  the  air,  through  the 
respiratory  tract,  skin  or  conjunctiva  or  through  ingestion  of  foods 
which  contain  the  specific  substance.  In  addition  to  air  contacts,  direct 
contacts  with  plants  and  animals,  which  may  or  may  not  serve  to  produce 
dusts,  may  also  lead  to  dermal  manifestations  of  hypersenstiveness. 

Serum  Disease. — The  Delayed  Reaction. — The  serum  treatment  of 
various  diseases  has  given  ample  opportunity  for  the  study  of  the 
symptom  complex  called  by  von  Pirquet  and  Schick  serum  disease. 
This  follows  with  extreme  frequency  upon  subcutaneous,  intravenous  or 
intrathecal  injections  of  animal  sera  employed  for  therapeutic  pur- 
poses and  may  be  delayed  or  accelerated.  The  symptoms  may  develop 
after  a  primary  or  series  of  primary  injections  and  constitute  the  delayed 
reaction.  These  symptoms  appear  from  six  to  twelve  days  after  the 
228 


HYPERSUSCEPTIBILITY  IN  MAN  229 

injection,  and  in  our  experience  have  been  most  frequent  after  ten  to 
eleven  days.  The  most  noticeable  and  most  common  symptom  is  a  skin 
eruption  which  is  usually  urticaria!  but  may  be  a  patchy  or  diffuse 
erythema,  a  scarlatiniform  or  a  multiform  eruption.  Edema  may 
appear  in  the  lips,  eyelids,  face  or  other  parts  of  the  body  and  rarely 
may  effect  the  larynx.  According  to  Longcope,  "  in  one  instance  a 
transient  hemiplegia  was  supposed  to  be  caused  by  local  edema  .of  the 
meninges."  We  have  seen  one  case  in  which  a  broncho-pneumonia, 
following  a  prophylactic  injection  of  serum  appeared  to  be  the  sequence 
of  an  edema  of  the  bronchi.  There  is  often  lymph-node  enlargement, 
which  may  precede  the  eruption  and  may  be  accompanied  by  enlarge- 
ment of  the  spleen.  There  is  likely  to  be  a  moderate  fever,  headache, 
malaise  and  occasionally  nausea  and  vomiting.  Multiple  joint  pains, 
increased  by  motion,  but  without  tenderness,  redness  or  swelling,  are 
common  in  severe  cases.  Albuminuria  appears  in  5  to  9  per  cent, 
of  the  cases,  and  Longcope  has  found  that  there  is  likely  to  be  salt 
and  water  retention  with  little  or  no  disturbance  of  nitrogenous  elim- 
ination. There  may  be  a  primary  leucocytosis,  followed  by  a  leuco- 
penia,  which  latter  shows  an  absolute  increase  of  lymphocytes.  The 
condition  usually  lasts  twenty-four,  forty-eight  or  seventy-two  hours 
and  occasionally  is  prolonged  to  twenty  days  or  more.  Relapses  may 
occur,  more  particularly  after  the  use  of  large  amounts  of  serum. 

Several  factors  enter  into  the  occurrence,  severity  and  duration  of 
the  disease,  the  larger  doses  giving  more  frequent  occurrence,  greater 
severity  and  longer  duration.  There  are  certainly  individual  differences 
in  the  resistance  of  patients  and  probably  individual  differences  in 
specimens  of  serum.  Sera  from  different  species  exhibit  differences 
in  toxicity  for  man,  that  of  the  ox,  according  to  Kraus,  being  less  likely 
to  produce  serum  disease  than  that  of  the  horse.  The  globulin  pre- 
cipitation or  so-called  concentration  of  horse  serum  in  the  preparation 
of  antitoxins  reduces  the  toxic  manifestations  in  man. 

The  Accelerated  Reaction. — Frequently  repeated  injections  of  serum 
at  properly  spaced  intervals  may  lead  to  a  state  of  resistance  or  im- 
munity, but  this  is  practically  never  permanent.  Following  a  primary 
injection  or  series  of  injections,  there  usually  develops  a  state  of  hyper- 
susceptibility.  This  condition  does  not  precede  the  appearance  of  the 
delayed  reaction  and  does  not  precede  the  tenth  day  after  injection, 
even  if  the  delayed  reaction  fails  to  appear.  Repeated  doses  of  serum  at 
short  intervals  delay  the  appearance  of  hypersusceptibility.  The  height 
of  sensitiveness  is  reached  in  from  two  to  three  months,  after  which  it 
slowly  subsides  but  probably  never  entirely  disappears.  We  have 
observed  accelerated  reactions  nine  and  fourteen  years  after  primary 
injection.  The  hypersusceptibility  exhibits  itself  only  on  injection  of 
the  protein  and  is  specific  for  the  species  from  which  it  originated. 
Following  the  second  injection  of  the  protein  or  serum  there  is  occa- 
sionally no  acceleration  of  reaction,  but  if  accelerated  it  may  be  mod- 
erately or  markedly  so,  the  last  producing  the  so-called  immediate 
reactions.  These  immediate  reactions  may  be  local,  appear  about  the  site 


230  THE  PRINCIPLES  OF  IMMUNOLOGY 

of  injection  in  from  a  few  minutes  to  an  hour  or  two  and  show  edema, 
erythema  or  urticaria.  There  may  also  be  a  general  immediate  reaction 
which  appears  in  from  twelve  to  twenty-four  hours  and  in  addition  to 
the  usual  symptoms  and  signs  of  serum  disease  may  be  accompanied 
by  severe  asthmatic  form  of  dyspnea,  cardio-vascular  disturbances  with 
cyanosis,  collapse,  chills,  nausea  and  vomiting  and  renal  disturbances 
including  complete  suppression  for  several  hours.  If  the  second  injection 
is  given  when  hypersusceptibility  is  not  marked,  as  for  example  after 
a  small  primary  dose,  or  several  years  after  a  primary  dose,  the  accel- 
erated reaction  is  not  likely  to  be  immediate  but  appears  in  from  two 
or  three  to  five  or  six  days.  Under  these  circumstances  the  reaction 
may  appear  as  an  ordinary  case  of  serum  disease  or  may  be  more  severe. 

Anaphy lactic  Shock  in  Man. — There  is  little  doubt  that  the  accel- 
erated reactions  of  serum  disease  bear  in  some  way  a  relation  to  ana- 
phylactic  shock.  During  the  period  of  hypersusceptibility  in  man  the 
subcutaneous  administration  of  serum  rarely  if  ever  produces  death, 
in  spite  of  the  fact  that  the  clinical  symptoms  may  be  extremely  severe. 
On  the  other  hand,  intravenous  injections  have  been  reported  to  produce 
death  following  symptoms  closely  resembling  those  of  anaphylactic 
shock  in  animals.  Reports  of  accidents  of  this  sort  led  to  the  funda- 
mental investigations  of  Rosenau  and  Anderson,  which  have  been 
described.  Injections  of  serum  into  the  spinal  canal  have  been  followed 
by  fatalities,  but  an  analysis  by  Auer  of  the  reported  cases  leads  him 
to  believe  that  for  the  most  part  these  deaths  were  due  to  other  causes 
than  anaphylaxis.  Miller  and  Root,  in  analysis  of  death  following 
subcutaneous  administration  of  horse  serum,  find  that  death  in  some 
instances  was  probably  caused  by  status  thymo-lymphaticus  and  that  in 
other  cases  the  cause  of  death  had  not  been  demonstrated  to  be  ana- 
phylactic. The  clinical  and  pathological  picture  of  fatalities  has  in  most 
instances  not  been  clearly  described.  Nevertheless,  Boughton  has 
recently  reported  a  case  in  which  a  man,  the  subject  of  bronchial 
asthma  when  near  horses,  died  upon  being  given  intravenously  one 
minim  of  normal  horse  serum,.  Autopsy  showed  enormous  distention 
of  the  lungs  with  congestion  of  other  viscera  and  numerous  small  hemor- 
rhages. This  apparently  is  an  instance  of  true  anaphylactic  shock  in 
man,  and  it  cannot  be  doubted  that  such  accidents  occur.  Caution 
must  be  exercised,  however,  in  attributing  death  to  anaphylaxis  because 
of  the  numerous  other  conditions  which  may  lead  to  sudden  death, 
particularly  in  the  course  of  acute  infectious  diseases. 

Natural  Hypersusceptibility. — The  recent  scientific  investigations 
of  hay  fever  and  its  various  modifications,  as  well  as  asthma,  eczema, 
other  diseases  of  the  skin,  angio-neurotic  edema  and  certain  gastro- 
intestinal disturbances,  have  shown  that  a  considerable  number  of  these 
cases  are  hypersusceptible  to  proteins  of  various  origins.  The  skin 
reactions,  to  be  described  subsequently,  and  the  effect  of  specific  treat- 
ment both  demonstrate  the  etiological  influence  of  the  special  proteins. 
The  evidence  presented  from  large  clinics  devoted  to  the  study  of  these 
conditions  leaves  no  doubt  concerning  the  fact  that  many  of  these  cases 


HYPERSUSCEPTIBILITY  IN  MAN  231 

are  instances  of  hypersusceptibility.  The  sensitive  state  appears  to  be 
inherent  in  the  cells  of  certain  individuals,  and  although  not  directly 
inherited,  Cooke  and  Van  der  Veer  have  found  that  the  tendency  to 
spontaneous  sensitization  appears  to  be  heritable,  that  it  follows  the 
law  of  Mendel  and  appears  as  a  dominant  character.  Nevertheless, 
there  is  a  possibility  that  sensitization  may  be  acquired  in  some  manner. 
Cooke,  Flood  and  Coca  maintain  that  artificial  sensitization  cannot  be 
produced  by  pollens.  Heyl,  however,  has  obtained  from  the  pollen  of 
ragweed  an  albumin,  a  proteose  and  a  globulin  and  found  that  mixtures 
of  the  albumin  and  proteose  possess  definite  sensitizing  properties  upon 
animal  inoculation.  Individuals  who  have  been  given  horse  serum 
therapeutically  become  somewhat  sensitive  to  subsequent  injections  of 
horse  serum,  but  only  in  rare  instances  is  the  sensitiveness  shown  as  a 
coryza  or  asthma  when  near  horses.  The  chance  of  sensitization  by  in- 
jection of  other  proteins  is  not  great.  The  possibility  of  sensitization 
by  virtue  of  the  material  gaining  access  to  the  body  through  the  respira- 
tory or  intestinal  surfaces  is  apparently  remote.  There  is  little  satis- 
factory evidence  that  protein  materials  in  the  form  of  dust  gain  access 
to  the  circulation  through  the  respiratory  membrane.  Ulrich,  however, 
reports  the  experimental  sensitization  of  guinea-pigs  by  nasal  insuffla- 
tion of  pollens  and  of  horse  serum,  but  reports  that  rabbits  cannot  be 
so  sensitized.  It  is  impossible,  under  these  circumstances,  to  exclude 
the  possibility  that  the  material  is  ultimately  swallowed,  and  sensitiza- 
tion effected  through  the  intestinal  tract.  The  ingestion  of  proteins  as 
foods  ordinarily  leads  to  such  changes  in  the  protein  in  the  process  of 
digestion  that  the  absorption  of  the  products  cannot  produce  sensitiza- 
tion. On  the  other  hand,  it  is  known  that  if  given  in  large  amounts  and 
given  under  certain  circumstances  native  protein  may  gain  access  to 
the  blood  stream  through  the  intestinal  tract.  Rosenau  and  Anderson 
maintained  that  sensitization  could  be  effected  by  feeding  horse  serum 
to  guinea-pigs,  but  the  failure  of  Besredka,  as  well  as  of  other  investi- 
gators, to  confirm  this  leaves  the  matter  in  some  doubt.  As  against  the 
acquisition  of  hypersusceptibility  in  hay  fever,  Dunbar  and  also  Cooke, 
Flood  and  Coca  have  found  that  patients  may  be  sensitive  to  the  pollens 
of  plants  indigenous  to  foreign  countries  and  with  which  the  patients 
have  never  come  in  contact. 

Hay  fever,  rose  fever  and  similar  disturbances  are  due  to  the  pol- 
lens of  certain  plants  and  the  flowering  period  of  these  plants  deter- 
mines the  seasonal  prevalence  of  the  disease.  The  pollens  responsible  are 
those  which  are  disseminated  by  winds ;  those  plants  which  are  pollin- 
ated by  insects  do  not  produce  hay  fever.  Scheppegrill  points  out 
also  that  the  direct  effects  of  pollens  are.  of  importance  as  they  may 
be  locally  irritant  to  both  normal  and  hypersusceptible  individuals, 
either  because  of  the  mechanical  effect  of  spiculated  pollens  or  because 
of  the  discharge  from  the  pollen  of  irritant  juices.  Local  reactions  may 
be  increased  by  anatomical  malformations  in  the  nose  and  pharynx,  such 
as  deviation  of  the  septum,  polyps,  adenoids,  and  the  condition  may 
entirely  subside  following  correction  of  these  abnormalities.  Strouse 


232  THE  PRINCIPLES  OF  IMMUNOLOGY 

and  Frank  claim  that  the  attacks  may  be  intensified  and  prolonged 
because  of  a  concurrent  acute  or  sub-acute  bacterial  infection,  which 
perhaps  permits  greater  absorption  of  the  pollen  protein.  The  condition 
may  be  so  severe  as  to  be  called  asthma,  and  in  addition  to  respiratory 
phenomena  may  show  erythematous  and  urticarial  eruptions.  Similar 
conditions  are  met  with  in  certain  individuals  sensitive  to  the  effluvia  of 
horses,  rabbits  and  other  animals.  It  is  well  known  that  the  ingestion 
of  certain  foods,  such  as  egg  albumin,  shell  fish,  strawberries,  may  give 
rise  to  serious  intestinal  disturbances  and  that  these  may  occasionally  be 
associated  with  skin  eruptions  or  respiratory  disturbance.  In  sensitive 
individuals  contact  of  the  skin  with  plants  or  animals,  to  the  protein  of 
which  the  individual  may  be  sensitive,  leads  not  uncommonly  to  cutane- 
ous eruptions.  These,  however,  are  not  likely  to  be  very  severe  or  of 
long  duration.  The  inflammation  of  the  skin  in  ivy  or  sumac  poisoning 
is  not  to  be  included  in  this  group,  because  the  irritant  agent  is  prob- 
ably not  of  protein  nature,  but  rather  an  acid-resin.  Eczema  and  per- 
haps certain  other  skin  diseases  may  also  be  due  to  hypersusceptibility, 
and  it  is  found  that  this  is  exhibited  rather  toward  food  products  than 
toward  other  forms  of  protein.  Furthermore,  certain  of  these  cases 
of  asthma,  eczema,  etc.,  may  be  due  to  bacterial  proteins  as  well  as 
those  of  higher  plants  and  of  animals. 

The  hypersusceptibility  of  the  sort  discussed  in  this  section  differs 
from  induced  hypersusceptibility  in  two  important  respects.  In  the 
first  place,  the  degree  of  sensitiveness  is  extreme.  This  may  be  illus- 
trated by  the  case  reported  by  Boughton,  quoted  above,  in  which  one 
minim  of  horse  serum  produced  death.  It  is  further  illustrated  by  the 
fact  that  hay  fever,  asthma  and  other  similar  conditions  are  induced 
by  what  must  necessarily  be  an  extremely  small  amount  of  protein 
in  the  atmosphere.  In  the  second  place,  the  sensitization  is  not  limited 
strictly  to  a  single  protein.  Longcope  classifies  these  individuals  roughly 
as  those  "  who  react  to  the  sera  of  animals ;  those  who  react  to  eggs, 
or  the  sera  of  fowls ;  those  who  react  to  the  extracts  of  shell  fish  and 
those  who  react  to  the  protein  of  plants."  Within  each  group  the 
individual  may  be  sensitive  to  the  protein  of  several  species.  As  has 
been  pointed  out  by  Walker,  those  who  react  to  bacteria  frequently 
react  to  several  varieties  of  organisms.  Furthermore,  individuals  may 
occasionally  show  reactions  to  two  or  three  of  the  large  groups  indicated 
by  Longcope.  Of  further  interest  in  regard  to  specificity  is  the  fact 
that  apparently  within  a  given  species,  proteins  of  somewhat  different 
origin  may  not  produce  identical  reactions.  For  example,  skin  icac- 
tions  may  demonstrate  sensitiveness  to  horse  dandruff  and  not  to  horse 
serum.  Desensitization  may  be  produced  by  careful  and  prolonged 
vaccination,  but  as  in  experimental  animals  the  desensitized  state  does 
not  persist  for  a  very  extended  period,  it  may  be  necessary  to  repeat 
the  vaccinations  every  six  months,  every  year,  or  at  such  other  periods 
as  the  individual  case  requires.  The  possibility  of  passive  sensitizition 
in  natural  hypersusceptibility  is  illustrated  by  a  case  reported  by 
Ramirez.  A  man  who  had  never  shown  any  hypersensitiveness  to  proteins 


HYPERSUSCEPTIBILITY  IN  MAN  233 

was  transfused  with  the  blood  of  a  donor  who  was  a  victim  of  horse 
asthma.  The  recipient,  two  weeks  later,  while  driving  in  a  carriage, 
was  seized  with  a  typical  asthmatic  attack  and  subsequently  showed  a 
positive  skin  reaction  to  horse  dandruff.  This  apparently  is  a  case  of 
passive  transfer  of  a  natural  hypersusceptibility.  No  data  have  been 
collected  to  show  whether  such  a  variety  of  passive  sensitization  is  per- 
manent in  man  or  exhibits  the  same  evanescent  character  as  occurs  in 
animals.  Passive  sensitization  of  animals  has  been  produced  by  Koess- 
ler,  but  Cooke,  Flood  and  Coca,  as  well  as  Ulrich,  have  been  unable 
to  confirm  this.  Our  own  experience  with  one  case  of  human  hyper- 
susceptibility  to  rabbit  serum  failed  to  demonstrate  passive  transfer 
into  guinea-pigs. 

Tests  for  Hypersusceptibility. — The  manifestations  of  hyper- 
susceptibility  may  be  general  or  lo'cal,  depending  on  the  mode  of  in- 
oculation and  the  amount  of  material  employed.  If  the  dose  can  be 
carefully  regulated,  the  hypersusceptible  state  may  be  demonstrated  by 
inducing  a  general  reaction,  as  in  the  tuberculin  reaction.  Owing  to  the 
fact  that  individuals  may  be  extremely  sensitive  to  certain  proteins,  as 
in  the  case  reported  by  Boughton  (see  page  230),  the  use  of  the  general 
reactions  for  diagnostic  purposes  is  limited  to  those  in  which  severe 
general  reactions  are  not  likely  to  appear.  The  local  reactions  give 
equally  satisfactory  information  in  man  and  are  devoid  of  serious 
results.  These  local  reactions  are  based  fundamentally  upon  the  studies 
of  Arthus,  published  in  1903.  He  found  that  if  animals  are  given 
several  subcutaneous  injections  of  normal  horse  serum  at  three-  or 
four-day  intervals,  the  first  three  injections  are  absorbed  readily,  but 
the  fourth  is  followed  by  a  local  inflammatory  reaction  and  subsequent 
injections  are  likely  to  be  followed  by  more  severe  inflammation,  necrosis 
and  gangrene.  Animals  rendered  sensitive  by  these  first  two  or  three 
injections  could  be  killed  by  intravenous  or  intraperitoneal  injections. 
The  Arthus  phenomenon  was  early  employed  as  a  means  of  detecting 
hypersusceptibility  resulting  from  bacterial  invasion,  but  it  has  now 
found  widespread  employment  in  the  detection  not  only  of  the  presence 
of  changes  incident  to  infectious  disease  but  also  for  the  determination 
of  sensitiveness  to  a  large  number  of  proteins  of  animal  and  vegetable 
origin.  Although  hypersusceptibility  may  exhibit  itself  in  respiratory 
phenomena  as  in  hay  fever,  horse  asthma,  etc.,  or  in  gastro-intestinal 
disturbance,  as  in  sensitiveness  to  egg-white,  definite  local  reactions 
may  be  evoked  by  the  introduction  of  the  proteins  into  or  under  the 
skki  and  these  local  reactions  may  be  accompanied  by  general  symp- 
toms, such  as  fever,  headache,  malaise  and  transitory  leucopenia  fol- 
lowed by  slight  leucocytosis  with  an  associated  esinophilia. 

Toxins  in  Hay  Fever. — The  studies  of  Dunbar  assumed  that  the  irri- 
tant agent  in  pollens  is  a  toxin.  He  based  this  conclusion  on  the  fact  that 
he  could  prepare  a  so-called  antitoxic  serum  "  pollantin  "  by  immunizing 
anjrnals  and  subsequently  claimed  that  he  could  demonstrate  antibodies 
by  precipitin  and  complement-fixation  tests.  Clowes  found  positive 
precipitation  and  complement  fixation  in  some  but  not  all  cases  before 


234  THE  PRINCIPLES  OF  IMMUNOLOGY 

the  beginning  of  the  hay-fever  season,  which  disappeared  for  a  few 
weeks  after  specific  desensitization.  On  the  other  hand,  numerous 
other  investigators  have  failed  to  demonstrate  such  reactions,  and  this 
phase  of  the  question  must  be  considered  unsettled.  Dunbar  claimed 
that  treatment  with  the  anti serum  "  pollantin  "  produced  specific  effects, 
but  Weichardt  maintains  that  equally  good  results  are  obtained  with 
the  serum  of  normal  animals  taken  in  the  summer  season.  Cooke, 
Flood  and  Coca  were  unable  to  demonstrate  immune  reactions  in  the 
sera  of  rabbits  inoculated  repeatedly  with  the  pollens  of  ragweed  and 
of  redtop.  Other  objections  to  the  toxin  conception  include  the  fact 
that  the  majority  of  normal  individuals  are,  practically  speaking,  abso- 
lutely resistant  to  the  pollens  and  fail  to  react  to  doses  1000  times  the 
dose  which  produces  reactions  in  susceptible  cases.  This  is  not  in 
accord  with  the  finding  in  regard  to  any  other  of  the  known  toxins. 
Apparently  normal  individuals  may  resist  diphtheria  toxin,  but  Cooke 
and  Van  der  Veer  have  pointed  out  that  such  resistance  depends  upon 
the  presence  of  demonstrable  antitoxin,  which  is  not  true  in  resistance 
to  pollens.  By  mixing  the  "  pollantin  "  and  pollens  and  then  testing 
by  an  ophthalmic  reaction  in  sensitive  individuals  Dunbar's  assistant, 
Prausnitz,  plotted  a  curve  of  neutralization,  but  Wolff-Eisner  found 
that  this  curve  does  not  follow  the  law  of  multiple  proportions  and  is 
therefore  not  similar  to  other  toxin-antitoxin  combinations.  There 
seems,  therefore,  little  ground  for  assuming  that  the  pollens  contain 
a  special  toxin  and  the  subsequent  work  with  hay  fever  and  similar 
conditions  indicates  that  they  represent  a  condition  of  hypersuscepti- 
bility  to  proteins  or  to  protein  decomposition  products. 

Technic  of  Cutaneous  Tests. — If  the  antigenic  proteins  are  already  in 
solution,  as  is  the  case  with  blood  serum,  no  especial  treatment  is  required 
other  than  suitable  dilution  under  strictly  aseptic  precautions.  If  the  protein 
is  in  solid  form,  as  in  the  case  of  vegetable  proteins  and  other  cellular  forms, 
extracts  are  required.  The  studies  of  Walker  and  of  Wodehouse  on  the 
preparation  of  materials  for  the  tests  have  been  of  the  utmost  importance. 
These  are  independent  of  the  preparation  of  the  various  tuberculins,  which 
will  be  discussed  subsequently.  They  found  that  an  excellent  dried  prepara- 
tion of  serum  could  be  obtained  by  precipitating  with  several  volumes  of 
acetone,  washing  the  precipitate  centrifugally  twice  with  alcohol  and  with 
ether,  and  drying  to  a  powder.  The  powder  may  be  applied  to  an  incision  in 
the  skin  and  dissolved  with  N/io  NaOH  solution.  Bacteria  are  cultivated  on 
solid  media,  washed  centrifugally  in  salt  solution,  then  twice  in  absolute  alcohol 
with  0.5  per  cent,  phenol  added,  twice  in  ether  and  then  dried  to  a  powder,  which 
may  be  used  as  is  the  serum  powder.  Cereals,  nuts  and  other  seeds,  roots 
and  tubers,  fruits,  leaves  and  stems  are  extracted  in  water,  precipitated  with 
95  per  cent,  alcohol,  washed  with  95  per  cent,  alcohol,  absolute  alcohol,  ether 
and  desiccated  over  hydrochloric  acid.  Hair  and  dandruff  of  animals  may 
be  employed  as  a  dissolved  extract  in  14  per  cent,  alcohol,  but  for  more 
accurate  studies,  dried  preparations  of  acid  metaprotein,  alkali  metaprotein 
and  pepton  extracted  from  the  material  are  employed. 

The  methods  of  inoculation  include  introduction  of  the  protein  into 
abraded  surfaces  and  intracutaneous  injection  through  a  fine  needle.  In 
special  instances,  as,  for  example,  in  the  use  of  tuberculin,  the  material  may 
be  incorporated  in  an  ointment  and  carefully  rubbed  into  the  skin;  this  is 
the  so-called  percutaneous  test.  Somewhat  similar  to  the  cutaneous  tests  is 
the  ophthalmo-reaction,  more  particularly  applied  in  tuberculin  tests,  where 
the  material  is  instilled  into  the  opnjunctival  sac.  Subcutaneous  injection  of 
material  is  also  resorted  to,  again  with  tuberculin  rather  than  with  other 


HYPERSUSCEPTIBILITY  IN  MAN  235 

substances,  but  the  determination  of  results  is  by  means  of  the  general 
rather  than  the  local  reaction.  As  with  other  reactions,  controls  are  a  neces- 
sary part  of  these  tests.  The  cutaneous  test,  by  which  is  meant  introduction 
of  material  into  an  abrasion,  is  performed  as  in  smallpox  vaccination.  Any 
part  of  the  body  may  be  selected,  but  we  have  found  the  arm  most  con- 
venient. Walker  advises  making  small  incisions  in  the  skin,  deep  enough  to 
permit  absorption,  but  not  deep  enough  to  cause  bleeding.  A  small  dental 
burr  may  be  used,  as  in  the  Schick  test.  The  material  is  placed  on  the 
abrasion  or  incision  and  allowed  to  remain  one-half  hour.  If  a  powder,  a 
solvent  should  be  added  after  the  powder  is  placed  on  the  skin.  If  not  com- 
pletely soluble  in  water,  a  weak  solution  of  sodium  hydroxide,  either  o.i  per 
cent,  or  N/io  may  be  employed,  as  it  does  not  affect  the  reaction.  Walker's 
studies  show  that  for  detecting  hypersensitiveness  in  cases  of  asthma,  hay 
fever,  etc.,  the  cutaneous  test  is  more  delicate  and  yields  fewer  false  positive 
reactions  than  the  intracutaneous  test. 

The  delicacy  of  these  tests  is  probably  greater  than  that  of  any 
other  biological  reaction.  As  has  been  stated,  patients  sensitive  to 
extracts  of  hair  of  an  animal  may  not  be  sensitive  to  the  serum  proteins 
and  vice  versa.  Very  small  amounts  of  antigen  suffice  to  produce  reac- 
tions; alkali  metaprotein  and  pepton  from  hair  and  dandruff  give 
reactions  commonly  in  dilutions  of  I— 10,000  and  Wodehouse  reports 
one  case  in  which  reactions  were  obtained  with  dilutions  of  1-1,000,000. 
Clowes  reports  reaction  by  means  of  the  ophthalmic  test  to  0.000,000,05 
gram  pollen.  The  fact  that  positive  reactions  are  found  with  cutaneous 
tests  in  individuals  whose  serum  fails  to  exhibit  antibodies  by  pre- 
cipitation, agglutination  and  complement-fixation  tests,  is  a  further 
indication  of  the  delicacy  of  the  reaction.  The  accuracy  of  the  reac- 
tions is  supported  by  the  .beneficial  results  of  specific  vaccination  or 
desensitization.  The  treatment  is  usually  by  means  of  subcutaneous 
injections  of  the  protein  to  which  the  patient  is  sensitive.  In  cases  of 
sensitiveness  to  food  products,  as  well  as  in  other  cases,  patients  may 
be  vaccinated  by  giving  the  protein  by  mouth.  In  either  method  the 
amounts  are  extremely  small,  and  in  most  instances  the  course  of  treat- 
ment must  be  repeated  at  intervals  which  may  vary  from  a  few  months 
to  a  year  or  more.  The  intracutaneous  test  appears  to  be  the  most 
delicate  in  producing  local  reactions,  but  unfortunately  is  more  likely 
to  produce  confusing  traumatic  and  non-specific  reactions  to  be  de- 
scribed subsequently.  Details  of  treatment  are  given  in  numerous 
articles,  such  as  those  of  Blackfan,  Talbot,  Goodale,  Berger  and  others 
in  the  recent  literature. 

The  Reaction. — This  depends  to  a  certain  degree  upon  the  particu- 
lar cutaneous  test  employed  and  the  sensitiveness  of  the  patient,  but  in 
a  general  way  the  description  applies  to  all  the  methods.  An  urticarial 
wheal  may  appear  within  a  very  few  minutes  and  may  persist  for  from 
several  minutes  to  several  hours,  elevated,  firm,  pale  and  itching.  Either 
with  or  without  this  preliminary  reaction,  the  passage  of  a  few  hours, 
six,  twelve,  twenty-four  or  more,  reveals  a  local  area  of  inflammation 
about  10  m.m.  in  diameter,  elevated,  papular,  red,  firm  and  tender.  In 
severe  reactions  the  area  may  reach  a  diameter  of  several  centimetres, 
may  be  surrounded  by  an  areola  of  subcutaneous  edema,  may  show 
fine  punctate  hemorrhages  and  may  ultimately  show  vesicles  and 


236  THE  PRINCIPLES  OF  IMMUNOLOGY 

crusts.  In  unusually  sensitive  individuals  the  local  reaction  may  be 
accompanied  by  systemic  manifestations.  Less  severe  but  sometimes 
confusing  reactions  may  appear  in  the  form  of  pseudo-reactions 
which  are  non-specific  in  nature  and  probably  due  to  the 
action  of  body  proteases  upon  introduced  proteins.  The  reaction 
to  the  traumatism  from  the  introduction  of  the  protein  may  at  times 
be  somewhat  confusing  but  in  most  instances  is  slight.  Certain  drugs, 
such  as  iodides  and  bromides,  appear  to  increase  the  intensity  of  reac- 
tions whether  they  be  specific  or  non-specific.  Iodides  are  known  to 
reduce  the  antiferment  titer  of  the  blood,  and  it  is  possible  that  the 
use  of  these  drugs  therefore  liberates  protease  and  in  this  way  acceler- 
ates the  non-specific  local  reaction.  The  increase  of  the  specific  local 
reactions  is  probably  due  to  the  increase  of  the  non-specific  interaction 
of  protease  and  the  introduced  protein. 

Theories  of  Cutaneous  Reactions. — The  appearance  of  local  reac- 
tions in  hypersusceptibility  may  be  explained  according  to  any  of  the 
theories  offered  for  anaphylactic  shock.  If  the  mechanism  of  ana- 
phylaxis  involves  the  formation  of  poisons  these  may  be  concentrated 
in  situ  because  of  the  irritation  produced  by  introducing  the  antigen. 
The  irritation  leads  to  a  slight  local  inflammation  with  its  incident  vaso- 
dilatation  and  edema.  Thus  there  is  a  local  concentration  of  antibody, 
which  in  reaction  with  the  introduced  antigen  produces  a  hypothetical 
poisonous  substance.  If  the  sensitizing  substance  is  within  cells,  the 
local  contact  of  antigen  in  the  tissues  of  the  skin  explains  the  local 
reaction.  Similarly  the  physical  theories  are  adaptable.  Stokes,  for 
example,  has  found  that  agar  will  produce  a  local  non-specific  reaction. 
This  is  probably  the  result  in  part  of  a  local  loss  of  balance  between 
ferment  and  antiferment  due  to  adsorption  of  the  latter  by  the  agar. 
Similarly  any  of  the  physical  theories  might  apply,  but  the  acceptance  of 
the  importance  of  the  cells  in  the  reaction,  whether  physical  or  other- 
wise, offers  an  excellent  reason  for  the  early  appearance  and  severity 
of  the  local  reaction  without  general  manifestations.  Cooke,  Flood  and 
Coca  state  that  antibodies  are  not  demonstrable  in  the  blood  of  naturally 
sensitive  persons  and  therefore  emphasize  the  essential  importance  of 
the  cells.  While  agreeing  that  the  cells  play  a  most  important  part, 
the  experiments  of  Koessler  and  the  case  reported  by  Ramirez  suggest 
that  natural  sensitization  is  of  essentially  the  same  nature  as  anaphylaxis, 
with  marked  differences  only  in  the  degree  of  cellular  and  humoral 
participation.  Therapeutic  desensitization  of  man  lasts  for  a  relatively 
short  period  of  time  and  differs  only  in  duration  from  desensitization 
in  experimental  animals.  In  both  cases  the  phenomenon  is  specific 
for  the  antigen  employed. 

Gay  and  Force,  Gay  and  Claypole,  and  Gay  and  Minaker,  in  their 
work  with  cutaneous  reactions  in  typhoid  fever  and  in  the  carrier  state 
in  meningococcus  infections,  have  expressed  the  opinion  that  positive 
reactions  are  an  indication  of  resistance  on  the  part  of  the  body  against 
infection  by  the  organisms  concerned.  Nichols  studied  the  typhoidin 
test  (see  page  242)  in  individuals  who  had  survived  typhoid  fever  and 


HYPERSUSCEPTIBILITY  IN  MAN  237 

found  that  only  75  per  cent,  of  these  reacted  positively,  whereas  experi- 
ence has  shown  that  at  least  90  per  cent,  of  such  individuals  are 
immune  to  reinfection.  He  also  pointed  out  that  the  immune  state 
following  an  attack  is  of  much  greater  duration  than  is  indicated  by  the 
typhoidin  test.  Furthermore,  those  who  have  survived  typhoid  fever  or 
have  been  vaccinated  with  bacillus  typhosus  react  positively  to  para- 
typhoidin,  but  it  is  known  that  these  individuals  are  not  immune  to 
paratyphoid  fever.  Kolmer  and  his  associates  have  found  no  con- 
stant parallelism  between  the  presence  of  positive  cutaneous  tests 
and  those  circulating  antibodies,  whose  presence  is  indicative  of  im- 
munity. "  The  positive  anaphylactic  skin  reaction  is,  therefore,  evidence 
of  infection  or  sensitization  to  a  particular  protein  without  bearing  any 
direct  relation  to  resistance  to  infection  or  reinfection." 

Drug  Idiosyncrasies. — It  is  well  known  in  connection  with  certain 
drugs,  such  as  morphin,  that  prolonged  use  makes  it  necessary  to  in- 
crease the  dose  in  order  to  obtain  physiological  effects.  This  increase  in 
resistance  to  morphin  is  specific,  but  in  the  case  of  chronic  alcoholism 
the  individual's  resistance  to  somewhat  related  substances,  such  as 
chloroform  and  ether,  is  also  increased.  Experiment  fails  to  show  that 
this  resistance  is  a  state  of  immunity,  and  no  immune  reactions 
in  the  ordinary  sense  of  the  term  have  been  demonstrated.  The  use 
of  certain  drugs,  such  as  iodof  orm,  iodides,  bromides,  coal-tar  products 
and  quinine,  sometimes  gives  evidence  on  the  part  of  the  patient  of 
a  special  susceptibility  or  idosyncrasy  in  the  form  of  cutaneous  erup- 
tions and  more  or  less  severe  general  symptoms.  Both  Bruck  and 
Klausner  expressed  the  view  that  this  is  an  evidence  of  hypersuscepti- 
bility  similar  to  or  identical  with  anaphylaxis.  Inasmuch  as  anaphylaxis 
is  a  phenomenon  concerning  proteins,  Bruck  offered  the  hypothesis 
that  the  drugs  enter  into  combination  with  the  body  proteins,  so  that 
a  new  drug-protein  complex  of  specific  character  is  formed.  This  pro- 
tein complex  may  act  as  a  sensitizer,  and  upon  subsequent  injection  of 
the  drug  there  occurs  a  combination  with  blood  proteins  to  produce 
a  similar  complex  which  reacts  with  the  sensitizer  to  produce  symptoms. 
Bruck  and  Klausner  claimed  to  be  able  to  sensitize  guinea-pigs  passively 
with  the  blood  of  susceptible  patients,  so  that  the  animals  reacted  with 
the  symptoms  of  anaphylaxis.  The  autopsies  on  these  animals  failed 
to  show  the  characteristic  findings  of  anaphylactic  shock.  Cole  studied 
patients  sensitive  to  iodides  and  to  copaiba  but  failed  to  obtain  results 
justifying  the  conclusion  that  the  phenomenon  should  be  included  among 
anaphylactic  manifestations.  Specific  cutaneous  reactions  to  such  drugs 
as  quinine  and  aspirin  have  been  described,  and  it  is  maintained  that 
small  doses  by  mouth  may  desensitize,  but  no  widespread  confirmation 
has  been  recorded.  None  of  the  drugs  studied  is  without  some  essential 
toxicity  and  the  idiosyncrasies  in  some  instances,  according  to  Sollmann, 
"  are  doubtless  due  to  differences  in  the  strength  or  constituents  of 
drugs."  He  further  states  in  regard  to  increased  susceptibility  that  it 
"  may  be  due  to  very  rapid  absorption,  or  slow  elimination ;  to  the 


238  THE  PRINCIPLES  OF  IMMUNOLOGY 

presence  of  synergistic  substances  in  the  body;  or  to  increased  func- 
tional susceptibility." 

The  Tuberculin  Test. — In  the  course  of  his  studies  on  the  treatment 
of  tuberculosis,  Koch  devised  the  method  of  diagnosis  which  we  now 
speak  of  as  the  general  tuberculin  reaction,  in  contrast  to  the  local 
reactions  subsequently  discovered.  It  is  now  known  that  the  introduc- 
tion of  tuberculin  into  the  body  may  lead  to  local  reactions  both  at  the 
site  of  inoculation  and  in  the  neighborhood  of  a  tuberculous  focus  as 
well  as  a  general  reaction  which  manifests  itself  in  fever,  headache 
and  malaise.  Numerous  methods  of  preparation  of  tuberculin  for 
therapeutic  and  diagnostic  purposes  have  been  described,  but  at  the 
present  time  the  diagnostic  methods,  in'  the  hands  of  the  majority  of 
workers,  depend  upon  the  use  of  original  or  old  tuberculin  of  Koch. 

For  the  preparation  of  this  tuberculin  now  designated  as  tuberculin  O.  T. 
the  organisms  are  grown  for  six  to  eight  weeks  on  the  surface  of  5  per  cent, 
alkaline  glycerine  broth  at  37°  C.  At  the  end  of  this  time  the  entire  contents 
of  the  flask  are  sterilized  and  concentrated  to  about  one-tenth  of  the  original 
volume  by  means  of  a  current  of  live  steam  and  a  water  bath.  The  glycerine 
does  not  evaporate,  and  as  a  result  of  the  concentration  constitutes  50  per  cent, 
of  the  final  mass.  This  is  filtered  through  porcelain  and  the  filtrate  employed. 
Koch  subsequently  made  other  preparations,  particularly  the  tuberculin 
known  as  T.  R.  and  that  known  as  B.  E.  The  T.  R.  or  tuberculin  residue  is 
prepared  by  growing  virulent  tubercle  bacilli  on  nutrient  glycerine  broth  for 
four  to  six  weeks  at  37°  C.  The  bacilli  are  obtained  by  filtration,  dried,  and 
ground  in  a  mortar.  One  gram  is  washed  with  100  c.c.  distilled  water,  the 
precipitate  is  again  dried,  powdered  and  repeatedly  washed  in  small  volumes 
of  water  until  no  sediment  results.  The  watery  extract  constituted  by  this 
second  series  of  washings,  which  should  not  exceed  100  c.c.,  is  preserved  with 
20  per  cent,  of  glycerine  and  constitutes  the  T.  R.  The  bacillus  emulsion 
(B.  E.)  is  prepared  by  growing  the  organisms  as  indicated  in  the  preparation 
of  the  original  or  old  tuberculin.  The  bacilli  are  obtained  by  filtration,  ground 
in  a  mortar  and  emulsified  in  100  parts  of  distilled  water  to  which  is  added  an 
equal  amount  of  glycerine. 

Numerous  other  methods  of  preparing  extracts  of  the  tubercle 
bacillus  have  been  described  but  are,  in  essential,  modifications  of 
the  methods  of  Koch.  At  the  present  time  the  original  or  old  tuber- 
culin is  used  most  widely. 

The  General  Reaction. — As  a  general  rule,  the  old  tuberculin  is  put  on 
the  market  in  the  form  of  ampoules  of  fluid,  i.o  c.c.  of  which  represents  i.o 
gram  of  pure  tuberculin.  This  may  be  diluted  for  the  actual  test.  Inasmuch 
as  individual  sensitiveness  varies  considerably,  the  primary  dose  should  be 
very  small.  According  to  Hamman  and  Wolman,  three  classes  of  patients 
may  be  recognized,  (a)  children,  (b)  patients  who  have  a  slight  fever  or  are 
not  in  good  general  condition,  (c)  patients  in  good  condition.  The  smaller 
doses  are  given  to  children  and  the  largest  dose  to  patients  in  good  general 
condition.  Upon  this  basis  the  initial  dose  of  old  tuberculin  should  be 
0.000,000,1  c.c.  to  0.000,001  c.c.;  failing  to  obtain  reactions  with  these  doses, 
subsequent  tests  may  be  made  at  intervals  of  about  a  week,  increasing  the 
dose  each  time.  Although  it  is  possible  to  give  a  maximal  dose  of  i.o  c.c.  of 
the  dilution,  it  is  rarely  advisable  to  exceed  0.05  c.c.  The  injections  should 
be  given  under  strict  aseptic  precautions,  and  appear  to  be  most  satisfactory  if 
given  at  the  lower  angle  of  the  scapula.  They  are  probably  best  given  in  the 
afternoon,  after  the  patient's  afternoon  temperature  has  been  taken,  so  as 
to  avoid  the  confusion  of  an  unusually  high  elevation  of  temperature  on  the 
day  selected.  The  reaction  appears  as  a  rule  in  from  twenty-four  to  thirty- 
six  hours.  It  may  appear  as  late  as  forty-eight  to  sixty  hours.  A  positive 
reaction  is  indicated  by  an  elevation  of  temperature  of  about  2°  to  4°  C.  In 


HYPERSUSCEPTIBILITY  IN  MAN  239 

addition  there  is  likely  to  be  headache,  malaise,  and  sometimes  a  loss  of 
weight.  At  the  site  of  inoculation  there  may  be  pain,  tenderness,  redness, 
swelling,  sometimes  associated  with  tenderness  and  enlargement  of  the 
regional  lymph  nodes.  The  contraindications  to  the  employment  of  the  test 
include  the  presence  of  fever,  if  fairly  high  and  continued,  nephritis,  gen- 
eralized miliary  tuberculosis,  intestinal  ulceration,  epilepsy,  acute  infectious 
diseases,  either  during  the  course  of  the  disease  or  its  convalescence. 

The  Cutaneous  Reaction. — Von  Pirquet,  who  first  described  this  modifi- 
cation of  the  tuberculin  test,  originally  recommended  the  use  of  25  per  cent, 
solution  of  the  old  tuberculin,  but  subsequently  found  that  the  undiluted 
material  is  more  suitable.  He  recommends  the  inner  (flexor)  surface  of  the 
forearm,  and  suggests  the  use  of  three  points  of  scarification  about  4  to  5  cm, 
apart.  The  skin  is  cleaned  with  ether  or  alcohol  before  making  the  abrasions. 
These  may  be  small  scratches  with  a  needle,  a  knife  or  with  an  instrument 
which  he  describes  as  a  borer,  which  has  a  sharp  chisel  point  and  is  rotated 
in  order  to  make  a  small  circular  abrasion.  A  drop  of  tuberculin  is  rubbed 
into  the  upper  and  lower  abrasions;  the  middle  one  remains  as  a  control.  In 
positive  cases,  the  reaction  about  the  point  of  inoculation  is  considerably 
greater  than  that  about  the  control  point.  The  traumatic  reaction  in  the 
control  may  reach  a  diameter  of  3  to  5  mm.  in  twenty-four  hours  and  then 
rapidly  disappears.  The  positive  reaction  usually  appears  within  twenty-four 
hours,  but  may  be  somewhat  delayed.  Its  diameter  is  ordinarily  about 
10  mm.,  but  may  reach  30  mm.  It  appears  as  a  red,  somewhat  tender  papule, 
which  in  severe  reactions  may  show  small  vesicles.  According  to  Kolmer,  it 
is  not  to  be  interpreted  as  positive  unless  its  diameter  exceeds  by  5  mm.  that 
of  the  control.  Occasionally  the  so-called  scrofulous  reaction  appears,  in 
which  papules  develop  upon  other  parts  of  the  extremities  and  the  trunk. 

The  Intracutaneous  Tuberculin  Tests. — This  was  described  by  Mendel 
and  also  by  Mantoux.  For  this  purpose  old  tuberculin  is  injected  into  the 
corium  in  doses  whose  bulk  is  0.05  c.c.  Two  injections  are  necessary,  one 
with  salt  solution  and  the  other  with  tuberculin.  The  injection  of  tuberculin, 
however,  may  include  three  doses  of  different  strengths.  The  reaction  is 
very  similar  to  that  of  the  cutaneous  test.  Following  a  subcutaneous  tuber- 
culin test,  a  similar  reaction  may  appear  in  the  track  of  the  needle. 

The  Percutaneous  Tuberculin  Test. — This  test  was  devised  by  Moro  and 
Doganoff  and  is  frequently  spoken  of  as  the  Moro  skin  test.  For  this  pur- 
pose 5.0  c.c.  of  old  tuberculin  are  thoroughly  mixed  with  5  grams  of  anhydrous 
lanolin.  This  may  be  preserved  for  a  long  time  in  a  light-proof  container  in 
the  refrigerator  and  may  be  obtained  on  the  market  in  collapsible  tubes. 
About  0.5  gram  of  this  ointment  is  rubbed  into  the  skin  of  the  abdomen,  or 
breast  near  the  nipple,  rather  vigorously  for  one  minute.  The  reaction  usually 
appears  within  twenty-four  hours,  but  may  be  delayed  from  four  to  six  days, 
and  it  subsides  in  three  to  ten  days.  It  usually  appears  as  a  number  of  small 
papules  reddened  at  the  base.  In  severe  cases  the  papules  may  become  con- 
fluent and  vesicles  may  form. 

The  Conjunctival  Tuberculin  Reaction. — This  is  also  referred  to  as  the 
ophthalmo-reaction  and  was  described  independently  by  Calmette  and  Wolff- 
Eisner.  Calmette  recommended  a  special  aqueous  extract  of  the  bacilli  but 
at  the  present  time  the  test  is  usually  applied  with  a  I  per  cent,  solution  of 
old  tuberculin.  One  drop  of  this  solution  is  instilled  into  the  conjunctiva 
near  the  inner  canthus.  The  opposite  eye  serves  as  a  control.  Even  in 
normal  individuals  the  instillation  may  induce  a  slight  reddening  of  the 
conjunctiva  within  six  hours,  but  the  positive  reaction  appears  in  from  six  to 
eight  hours,  reaches  its  height  in  from  twenty-four  to  forty-eight  hours,  and 
then  subsides  in  a  few  days  or  a  week.  The  reaction  may  include  simply  a 
slight  reddening  and  swelling  of  the  caruncle,  including  the  neighboring 
part  of  the  lower  lid,  may  extend  over  the  scleral  conjunctiva  or  may  lead 
to  a  purulent  conjunctivitis.  Following  the  introduction  of  this  test,  unfavor- 
able reports  were  made  because  of  the  seeming  danger  of  producing  perma- 
nent injury  to  the  eye,  but  Hamman  and  Wolman  state  that  this  danger  is 
not  considerable,  provided  proper  precautions  are  taken  in  the  selection  of 
patients.  Diseases  of  the  eye  or  of  the  skin  near  the  eye,  obvious  scrofula  in 
children,  and  arterio-sclerosis  are  contraindications.  The  test  should  never 
be  applied  twice  in  the  same  eye,  and  no  stronger  solution  than  I  per  cent, 
should  be  employed  for  the  first  test.  If  the  first  test  is  negative  a  5  per  cent. 
solution  may  subsequently  be  employed  in  the  opposite  eye. 


24o  THE  PRINCIPLES  OF  IMMUNOLOGY 

Theories  of  the  Tuberculin  Reaction. — Koch  is  of  the  opinion  that 
the  amount  of  tuberculin  introduced  when  added  to  that  already  present 
in  the  body  provides  a  .sufficient  amount  of  toxic  substance  to  produce 
a  definite  general  reaction.  Koehler  and  Westphal  thought  that  a 
toxic  body  is  formed  in  the  tuberculous  focus  by  the  union  of  tuberculin 
and  the  products  of  the  tubercle  bacillus.  Marmorek  suggested  that 
the  tuberculin  excited  the  tubercle  bacilli  to  produce  in  excess  those  toxic 
bodies  which  lead  to  fever.  Von  Pirquet  and  Schick  were  the  first  to 
suggest  that  this  is  a  phenomenon  related  to  hypersusceptibility.  This 
conception  fits  very  well  the  view  of  the  relation  of  anaphylaxis  and 
immunity  which  we  have  indicated  above  (page  226).  Individuals  who 
have  markedly  active  tuberculosis  are  not  likely  to  react,  whereas  those 
who  have  quiescent  or  cicatrized  lesions  almost  always  react.  If  the 
presence  of  tuberculosis  leads  to  the  formation  of  a  sensitizing  sub- 
stance, this  can  well  be  absorbed  by  the  cells  and  be  responsible  for  the 
local  and  general  reactions.  The  tuberculin,  upon  local  application, 
may  react  with  a  sensitizing  substance  in  the  situation  concerned,  or 
upon  entrance  into  the  circulation  may  similarly  react  with  the  sensitiz- 
ing substance  in  more  widely  distributed  cells,  thus  producing  a  general 
reaction  similar  in  principle  to  anaphylactic  shock.  If,  on  the  other 
hand,  the  tuberculous  process  is  so  active  that  immune  bodies  can  be 
found  in  the  circulating  blood,  combination  may  be  effected  in  that  situ- 
ation and  the  cells  protected.  The  study  of  complement  fixation  in 
tuberculosis  indicates  that  this  latter  assumption  is  true,  namely,  that 
those  who  have  active  tuberculosis  are  more  likely  to  react  positively 
by  the  complement-fixation  test,  thereby  indicating  the  presence  of  cir- 
culating antibodies  in  the  active  stages  of  the  disease. 

Krause  has  studied  this  problem  extensively,  particularly  in  experi- 
mental animals  and  finds  no  reason  for  associating  skin  hypersensitive- 
ness  and  anaphylaxis.  The  anaphylactic  state  may  be  induced  in 
animals  by  parenteral  injection  of  tuberculo-protein,  but  they  do  not 
acquire  cutaneous  hypersensitiveness.  Only  by  establishing  a  focus 
of  infection,  is  it  possible  to  demonstrate  a  skin  reaction.  Although 
during  the  period  of  anaphylactic  shock  an  animal  may  appear  to  be 
somewhat  less  resistant  to  infection,  the  state  of  anaphylaxis  produces 
no  alteration  in  its  resistance.  Krause  is  of  the  opinion  that  tissue  and 
cutaneous  hypersensitiveness' and  immunity  to  infection  occur  under 
the  same  conditions,  and  that  one  may  probably  be  a  function  of  the 
other.  In  the  experimental  animal  the  degree  of  cutaneous  hyper- 
susceptibility  and  immunity  parallel  each  other.  He  suggests  that  the 
local  reaction  may  also  appear  in  the  neighborhood  of  foci  of  infection 
and  thus  aid  in  walling  off  the  infecting  agent.  Krause's  opinion, 
based  on  much  admirable  work,  is  worthy  of  the  highest  consideration, 
but  in  so  far  as  we  can  determine,  it  is  not  in  accord  with  studies  of 
immunity  and  cutaneous  reactions  in  many  other  conditions,  as  pointed 
out  in  our  discussion  of  cutaneous  hypersusceptibility  in  general.  Peter- 
sen  considers  the  tuberculin  reaction  as  a  two-phase  phenomenon.  The 
primary  alteration  of  the  ferment-antiferment  balance  brings  about  a 


HYPERSUSCEPTIBILITY  IN  MAN  241 

medium  favorable  for  proteolysis  in  and  about  the  tubercle.  Digestion 
and  the  liberation  of  toxic  material  result  and  are  reflected  in  the  con- 
stitutional effects.  In  the  non-tuberculous  individual  it  is  probable 
that  the  primary  serum  alterations  also  occur,  but  the  digestive  ferments, 
finding  no  focus  to  attack,  liberate  no  toxic  material  and  no  general 
reaction  is  elicited.  Any  agent  that  brings  about  a  f  erment-antif  erment 
ratio  favorable  for  proteolysis  will  effect  a  general  reaction  provided 
the  focus  be  sufficiently  unstable.  Conditions  such  as  pregnancy,  acute 
infections,  protein  shock,  in  which  there  is  an  increase  of  antif erment, 
will  inhibit  the  reaction.  In  late  stages  of  tuberculosis  there  is  also 
increased  antiferment  and  therefore  less  marked  local  reactions  but 
more  marked  general  reactions. 

Specificity  of  the  Tuberculin  Reaction. — The  tuberculin  tests  have 
probably  been  more  carefully  controlled  by  autopsy  than  any  of  the 
other  clinical  tests,  and  we  therefore  are  able  to  state  with  considerable 
assurance  that  a  positive  reaction  indicates  the  presence  of  tuberculosis 
in  the  vast  majority  of  cases,  but,  on  the  other  hand,  gives  no  very 
precise  information  as  to  the  degree  of  activity  of  the  process.  Factors 
of  error  are  more  particularly  found  in  the  personal  equation  of  the 
examiner.  Leprosy  and  actinomycosis,  however,  may  give  confusing 
results.  In  a  very  large  series  of  tests,  more  than  15,000,  the  percentage 
of  error  is  very  small,  varying  from  2  to  3  per  cent.  Negative  reactions 
may  appear  in  markedly  active  tuberculosis,  in  the  very  early  stages  of 
the  infection,  in  those  small  cicatrized  lesions  of  the  lung  so  firmly 
encapsulated  that  no  absorption  takes  place,  during  continued  treatment 
with  tuberculin ;  also  during  the  course  of  measles,  typhoid  fever, 
acute  articular  rheumatism,  pneumonia,  diphtheria,  pertussis,  serum 
disease  and  during  pregnancy. 

Some  authors  have  such  confidence  in  the  specificity  of  the  tuber- 
culin reaction  that  they  consider  it  possible  to  determine  the  strain  of 
the  organism  concerned,  but  others  deny  that  this  delicacy  is  attainable. 
The  recent  work  of  Petersen  would  indicate  that  there  is  a  large  non- 
specific element  in  the  tuberculin  reaction.  Tuberculous  patients  may 
react  to  the  following  substances  with  local  and  even  general  reactions : 
hypertonic  salt  solution,  distilled  water,  iodides,  some  colloidal  metals, 
protein  split  products,  etc.  Non-tuberculous  individuals  will  tolerate 
equal  doses  without  reaction.  The  relation  of  this  type  of  reaction  to 
the  true  test  has  been  indicated  in  discussion  of  the  theories  of  the 
tuberculin  test. 

Utility  of  the  Tests. — At  the  present  time  in  clinical  practice  the 
subcutaneous  or  general  reaction  is  not  very  widely  employed,  because 
of  the  prejudice  that  has  been  aroused  by  the  possibility  of  exciting  the 
lesion  to  renewed  activity.  Similarly  a  prejudice  exists  somewhat 
unjustly  against  the  use  of  the  conjunctival  reaction.  Although  Ham- 
man  and  Wolman  indicate  that  the  intracutaneous  test  is  the  most  sensi- 
tive, our  observation  is  to  the  effect  that  the  cutaneous  test  is  most 
widely  employed.  It  is  simple,  free  from  danger,  well  controlled, 
16 


242  THE  PRINCIPLES  OF  IMMUNOLOGY 

easily  read  and  is  sufficiently  sensitive  to  provide  all  the  information  that 
can  reasonably  be  expected  to  accrue  from  the  tuberculin  test. 

Luetin  Reaction. — Numerous  attempts  were  made  following  the 
announcements  of  the  Von  Pirquet  cutaneous  tuberculin  test,  to  devise 
a  similar  test  for  syphilis.  It  was  found,  however,  that  extracts  of 
normal  organs  produced  the  same  effects  as  those  from  syphilitic  organs. 
It  was  not  until  Noguchi  cultivated  the  treponema  pallidum  in  -vitro  that 
a  preparation  of  the  causative  agent  could  be  prepared.  Noguchi  pre- 
pared a  suspension  of  the  organisms  together  with  the  ascites-kidney 
agar  upon  which  they  were  grown.  Cutaneous  reactions  were  unsat- 
isfactory, and  it  was  found  necessary  to  make  the  test  by  intracutaneous 
injection  of  the  material.  The  reaction  appears  in  papular  or  pustular 
form  in  from  twenty-four  to  forty-eight  hours  or  later.  It  was  found 
by  Sherrick  that  patients  receiving  potassium  iodide  give  positive  reac- 
tions and  by  Cole  and  Paryzek  that  similar  reactions  follow  the  ad- 
ministration of  bromides.  Although  Noguchi  found  that  injection  of 
the  culture  medium  without  the  organisms  did  not  produce  reactions, 
Stokes  as  well  as  Kolmer,  Matsunami  and  Broadwell  were  able  to  pro- 
duce reactions  by  injecting  agar.  Although  Noguchi  and  others  re- 
ported high  percentages  of  positive  reactions  in  known  syphilitics,  yet 
in  the  hands  of  some  workers  the  number  has  been  only  about  50  per 
cent.  The  test  is  not  widely  employed  and  apparently  gives  no  informa- 
tion that  cannot  be  obtained  equally  well  or  better  from  the  Wasser- 
mann  test.  It  has  been  suggested  that  the  luetin  test  may  be  of  value 
in  late  syphilis,  where  the  Wassermann  test  is  negative,  but  the  large 
non-specific  element  of  this  skin  reaction  does  not  tend  to  place  much 
reliance  upon  the  test 

Cutaneous  Reactions  in  Typhoid  Fever. — Several  of  the  earlier 
studies  on  this  subject  were  concerned  with  reactions  in  the  conjunctiva. 
Chatemesse  and  also  Austrian  were  able  to  obtain  positive  ophthalmo- 
reactions  in  a  large  percentage  of  cases  of  typhoid  fever  and  prac- 
tically no  positive  reactions  in  other  individuals.  Although  Kraus  could 
not  obtain  skin  reactions,  Zupnik  and  also  Floyd  and  Barker  obtained 
encouraging  results.  Gay  and  Force  have  employed  a  substance  which 
they  name  typhoidin,  prepared  from  bacillus  typhosus,  according  to  the 
method  employed  for  the  preparation  of  old  tuberculin.  The  prepara- 
tion was  modified  subsequently  by  Gay  and  Claypole.  The  typhoidin 
is  applied  in  abrasions  of  the  skin  as  with  the  cutaneous  tuberculin  test. 
These  investigators  found  a  high  percentage  of  positive  reactions  in 
individuals  who  had  recovered  from  typhoid  fever  as  well  as  those 
who  had  been  vaccinated  and  recommend  it  as  a  method  for  determining 
the  presence  of  immunity  to  typhoid  fever.  Kilgore  has  studied  the 
test  clinically  and  finds  that  the  test  is  unreliable  because  of  unavoidable 
variations  in  the  application  of  the  test,  indefiniteness  of  the  readings 
and  the  large  non-specific  element  in  the  reaction. 

Cutaneous  Reactions  to  Gonococcus  Infections. — These  reactions 
are  particularly  applicable  to  deep-seated  and  chronic  infections  with 
the  gonococcus.  Irons  found  local  and  general  reactions  following  the 


HYPERSUSCEPTIBILITY  IN  MAN  243 

subcutaneous  injection  of  gonococcal  vaccine  and  subsequently  pre- 
pared a  glycerol  extract  of  the  organism  for  cutaneous  tests.  He 
instituted  controls  with  equal  quantities  of  glycerol  and  obtained  dis- 
tinctly encouraging  results  even  to  the  point  where  one  strain  of  or- 
ganism produced  stronger  reactions  than  other  strains. 

Cutaneous  Reactions  to  Meningococcus  Infections. — Recently  Gay 
and  Minaker  have  employed  the  intracutaneous  reaction  for  the  detec- 
tion of  meningococcus  carriers.  They  prepared  a  salt  solution  emulsion 
of  carefully  washed  and  thoroughly  dried  cultures  of  five  strains  of 
meningococcus  and  injected  0.000,006  gram  of  the  dried  powder  in  a 
total  volume  of  0.05  c.c.  They  obtained  reactions  in  64.5  per  cent,  of 
known  carriers  and  26.4  per  cent,  in  individuals  known  not  to  be 
carriers.  They  do  not  think  that  the  reaction  serves  any  important 
purpose  in  diagnosis  but  suggest  that  it  may  indicate  a  systemic  reaction 
and  possibly  a  certain  degree  of  acquired  resistance  to  the  organism. 

Cutaneous  Reactions  to  Pneumococcus  Infections. — Earlier  in- 
vestigations with  salt-solution  extracts  were  not  particularly  satis- 
factory in  regard  to  the  early  diagnosis  of  pneumonia,  although  after 
the  crisis  reactions  were  obtained.  Weiss  and  Kolmer  prepared  a 
solution  of  Type  I  pneumococci  in  sodium  choleate  which  they  designate 
pneumotoxin.  The  test  is  performed  by  intracutaneous  injection.  By 
careful  study  of  animals,  on  the  basis  of  both  gross  and  microscopic 
examination  of  the  site  of  reaction,  as  well  as  of  human  patients  with 
pneumonia,  they  obtained  distinctly  encouraging  results  during  the 
course  of  the  disease  and  state  that  although  the  test  does  not  seem 
to  be  of  distinct  value  in  differentiating  the  type  of  organism  con- 
cerned, yet  it  may  aid  in  differential  diagnosis  between  appendicitis, 
tuberculosis  and  pneumonia. 

Cutaneous  Reactions  to  Vaccine  Virus. — Jenner  noted  that  in  cer- 
tain individuals  who  had  previously  been  vaccinated  against  smallpox, 
a  second  vaccination  might  produce  a  local  reaction  which  did  not  go  on 
to  produce  vaccinia.  This  has  been  observed  by  numerous  investi- 
gators since  then  and  Force  has  given  the  subject  close  study.  For 
this  purpose  Force  produced  three  abrasions  on  the  arm,  into  two  of 
which  vaccine  virus  was  rubbed  and  made  observations  at  the  end  of 
twenty-four,  forty-eight  and  seventy-two  hours.  "  If  either  of  the  vac- 
cinated spots  showed  an  areola  of  5  mm.  or  over  (with  or  without 
papule)  at  the  end  of  twenty-four  hours,  which  areola  (or  papule)  had 
decreased  at  the  tim-e  of  the  seventy-two-hour  observation,  it  was  con- 
sidered a  reaction  of  immunity  due  to  the  presence  in  the  blood  of  the 
individual  of  antibodies  against  vaccine  virus."  "  If  either  of  the  vac- 
cinated spots  showed  an  areola  at  the  end  of  twenty-four  hours  which 
developed  into  a  small  vesicle,  maturing  on  the  fifth  or  sixth  day  and 
then  rapidly  subsiding  the  reaction  was  considered  a  v&ccinoid"  a  con- 
dition in  which  it  is  supposed  antibodies  are  not  present  but  are  rapidly 
formed  because  of  a  previous  vaccination,  thus  leading  to  the  small 
size  and  rapid  subsidence  of  the  vesicle.  "  If  there  was  no  change  until 
the  third  day,  and  then  a  small  areola  began  <to  form,  the  case  would  be 


244  THE  PRINCIPLES  OF  IMMUNOLOGY 

vaccinia"  This  description  indicates  the  changes  that  may  appear  fol- 
lowing an  uninfected  vaccination  with  smallpox  virus.  There  appar- 
ently occurs,  following  smallpox  and  vaccinia,  an  altered  state  which 
determines  these  local  reactions,  but  the  interpretation  offered  by  Force 
that  some  of  these  reactions  are  immune  reactions  still  lacks  satis- 
factory confirmation  and  is  not  consistent  with  other  studies  of 
cutaneous  reactions  (see  page  237). 

Cutaneous  Reactions  in  Glanders. — The  Mallein  test  devised  by 
Kelmann  and  Kelming  is  widely  employed  in  veterinary  practice,  either 
in  the  form  of  subcutaneous  injection  which  produces  a  general  reaction 
as  is  the  case  with  tuberculin,  or  in  the  form  of  conjunctiva!  test  which 
produces  local  and  often  general  reactions. 

Other  Cutaneous  Reactions. — As  can  very  well  be  understood  the 
encouraging  results  with  such  a  large  number  of  skin  reactions  has 
led  to  the  investigation  of  similar  tests  in  other  diseases  and  the  reac- 
tion has  been  applied  in  leprosy,  sporotrichosis,  hyphomycetes  infec- 
tions, pregnancy,  canine  distemper  and  numerous  other  conditions. 
The  Schick  test  for  diphtheria  is  not  to  be  included  among  the  skin 
reactions  indicating  hypersusceptibility,  for,  as  has  been  shown  pre- 
viously, this  test  depends  upon  the  presence  or  absence  of  antitoxin  in 
the  circulating  fluids  of  the  body. 


CHAPTER  XII 
DEFENSIVE  FERMENTS 


INTRODUCTION. 
SPECIFICITY   OF    FERMENTS. 
IMMUNE  FERMENTS. 
FERMENTS  IN  THE  BLOOD. 

FERMENT-ANTIFERMENT  BALANCE. 

ANTIFERMENT. 

THE  ABDERHALDEN  TEST. 


Introduction. — The  relation  of  ferments  to  immunity  and  ana- 
phylaxis  has  long  been  the  subject  of  discussion.  In  the  chapters  on 
special  immune  bodies  we  have  discussed  the  similarities  and  differences 
between  ferments  or  enzymes  and  antibodies.  Special  consideration  has 
been  given  to  certain  phases  of  ferment  activity,  particularly  in  the 
chapter  on  Cellular  Resistance  and  that  on  Hypersusceptibility.  Ap- 
parently the  first  work  to  prove  that  digestion  takes  place  outside  the 
intestinal  tract  was  that  of  Hammersten,  who  showed  in  1885  that 
washed  leucocytes  increase  the  solubility  of  fibrin.  This  was  followed 
by  more  comprehensive  studies  on  cellular  ferments  as  have  been  pre- 
viously outlined  (page  167).  In  addition  to  those  ferments  which  exist 
in  the  cells,  ferments  have  been  discovered  in  the  blood  and  other  circu- 
lating body  fluids.  Therefore,  we  may  classify  the  ferments  as  intra- 
cellular  and  extracellular.  The  scope  of  this  book  is  too  limited  to 
permit  of  any  general  discussion  of  ferments  as  a  group  and  the 
reader  is  referred  to  the  sections  on  this  subject  in  Wells'  "  Chemical 
Pathology."  Many  of  the  earlier  workers  assumed  that  ferments  in 
the  body  fluids  are  derived  essentially  from  the  leucocytes.  A  recent  study 
of  considerable  significance  is  that  of  Boldyreff.  He  maintains  that  the 
glands  of  the  alimentary  canal,  with  the  exception  of  those  of  the 
3tomach,  are  not  at  rest  between  the  digestive  periods  and  that  they 
exhibit  a  periodic  function.  As  a  result  of  this  periodicity,  secretions 
are  discharged  into  the  empty  intestine  from  which  they  are  absorbed 
and  at  times  are  demonstrable  in  the  blood.  Van  Calcar  claims  that 
the  leucocytes  are  incapable  of  producing  their  own  ferments  and  that 
these  ferments  are  derived  from  special  glands.  He  found  that  extir- 
pation of  the  stomach  is  followed  by  a  decrease  or  absence  of  that 
ferment  of  the  leucocytes  which  acts  best  in  acid  medium  and  that 
extirpation  of  the  pancreas  similarly  is  followed  by  a  loss  of  tryptic 
powers  on  the  part  of  the  leucocytes.  Abderhalden  believes  that  invertin 
also  is  derived  from  the  intestinal  glands.  This  conception  would  indi- 
cate that  the  appearance  of  ferments  in  the  circulating  body  fluids  is  to 
be  regarded  as  a  mobilization  of  ferments  from  the  cells  which 
formed  them. 

Specificity  of  Ferments. — It  is  well  known  that  the  body  ferments 
act  specifically  upon  certain  chemical  substances,  as  exemplified  by  the 

245 


246  THE  PRINCIPLES  OF  IMMUNOLOGY 

digestion  of  starch  by  amylase  and  of  protein  by  pepsin.  The  question 
as  to  whether  or  not  specificity  in  the  immunological  sense  can  be  demon- 
strated has  been  the  subject  of  much  discussion.  Claims  have  been 
made  for  specificity  of  ferments  not  only  in  regard  to  animal  species 
but  also  in  regard  to  specificity  for  the  cells  of  particular  organs.  The 
chief  proponent  of  the  specificity  of  ferments  for  cells  and  proteins  is 
Abderhalden.  He  was  stimulated  to  this  view  by  the  work  of  Schmorl 
and  others,  who  demonstrated  that  during  pregnancy  fragments  of  the 
syncytium  of  the  chorionic  villi  often  enter  the  circulation  and  by  the 
claims  of  Weinland  that  specific  reducing  ferments  are  produced  fol- 
lowing the  parenteral  introduction  of  cane  sugar.  Abderhalden  there- 
upon examined  the  blood  serum  of  pregnant  animals  and  found  that 
the  serum  contained  a  ferment  capable  of  splitting  placental  pepton 
into  amino-acids  and  of  digesting  coagulated  placental  tissue  into  pep- 
ton,  polypeptids  and  amino-acids.  These  decomposition  products  are 
diffusible  and  also  alter  the  axis  of  optical  rotation  of  the  mixture.  The 
detection  of  the  diffusible  products  of  protein  decomposition  was  made 
by  means  of  "  ninhydrin  "  or  triketohydrindenhydrate,  which  reacts 
with  alpha  amino-acids  so  as  to  produce  a  blue  or  violet  color.  The 
practical  application  of  a  test  of  this  sort  is  obvious  and  the  method 
has  been  employed  to  detect  specific  ferments  in  pregnancy,  in  car- 
cinoma, in  sarcoma,  in  diseases  o/f  the  brain,  of  the  eye  and  of  numerous 
other  organs.  Practical  experience  with  the  test,  as  well  as  further 
scientific  study,  has  made  it  seem  probable  that  the  specificity  claimed 
by  the  Abderhalden  school  does  not  exist.  This  will  be  further  dis- 
cussed in  connection  with  the  Abderhalden  test. 

Immune  Ferments. — Numerous  investigators  have  published  re- 
ports indicating  that  the  parenteral  introduction  of  special  sub- 
stances leads  to  production  of  special  ferments  or  at  least  to  an  increase 
of  preexisting  ferments  in  the  form  of  a  mobilization.  Delezenne  re- 
ported in  1900  that  the  injection  of  animals  with  gelatine  produces  a 
blood  serum  capable  of  liquefying  gelatine.  Weinland  in  1907  showed 
that  although  normal  dog  serum  cannot  reduce  cane  sugar  the  immuniza- 
tion of  a  dog  by  several  injections  of  cane  sugar  leads  to  the  formation 
of  a  ferment  capable  of  reducing  cane  sugar  in  vitro.  Similarly,  im- 
munization with  edestin  produces  a  serum  capable  of  splitting  this 
substance.  The  more  recent  investigations  of  the  subject  would  make 
it  appear  that  the  immunization  leads  rather  to  mobilization  of  non- 
specific ferments  than  to  the  production  of  a  specific  immune  body. 

Ferments  in  the  Blood. — Wells  states  that  the  blood  contains  di- 
astase, glucase,  lipase,  thrombin,  rennin  and  proteases.  In  addition, 
the  blood  possesses  oxidizing  properties  due  presumably  to  the  presence 
of  oxydase,  peroxydase  and  probably  also  due  to  catalase.  The  pro- 
teases have  been  given  particularly  careful  study.  Petersen  divides 
these  ferments  into  the  leucoproteases,  serum  proteases  and  serum  pep- 
tidases.  The  leucoproteases  include  (a)  an  active  ferment  operating  in 
alkaline  medium  and  capable  of  digesting  native  protein  to  the  proteose 
stage,  (b)  an  active  ferment  capable  of  operating  in  acid  medium  with 


DEFENSIVE  FERMENTS  247 

a  similar  range  of  activity,  and  (c)  an  ereptase  active  in  both  acid  and 
alkaline  media  and  capable  of  splitting  partially  hydrolized  proteins  into 
amino-acids.  Of  these  ferments  only  the  ereptase  is  able  to  act  in  the 
presence  of  blood  serum  and  tissue  fluids  because  the  others  are  inhibited 
by  the  activity  of  antiferment  constantly  present.  The  serum  protease 
is  a  polyvalent  trypsin-like  ferment  active  in  neutral,  in  slightly  acid, 
or  in  slightly  alkaline  media ;  it  is  completely  inhibited  by  the  antifer- 
ment of  the  circulating  fluid.  It  becomes  active  only  when  the  anti- 
ferment  is  removed  and  is  capable  of  digesting  native  protein  to  the 
amino-acid  stage.  It  is  present  in  fairly  large  amounts  in  sera  of  the 
lower  animals,  but  is  found  in  only  small  quantities  in  human  serum. 
The  serum  peptidase  is  a  polyvalent  ferment  which  operates  in  the 
same  type  of  media  as  the  protease ;  it  is  present  in  normal  human  serum 
in  small  amounts,  is  not  inhibited  by  antiferment  and  digests  partly 
hydrolized  proteins  to  the  amino-acid  stage.  Since  the  toxic  fractions 
of  proteins  are  principally  in  the  form  of  proteoses  and  pepton,  the 
peptidase  apparently  is  the  most  important  ferment  in  destroying  such 
toxic  bodies. 

The  importance  of  esterases  in  the  blood  is  not  at  all  clear.  It  is 
known  that  lymphocytes  contain  a  lipase,  and  it  has  been  suggested 
that  the  accumulation-  of  these  cells  about  tuberculous  foci  may  indicate 
the  importance  of  this  ferment  in  breaking  down  the  waxy  capsule  of 
the  bacilli.  Jobling,  Petersen  and  Eggstein  recommend  the  following 
methods  for  the  determination  of  serum  protease  and  serum  esterase 
(Journal  of  Experimental  Medicine,  vol.  xxii.). 

"  The  technic  for  proteases  is  as  follows :  The  clear  hemoglobin-free  serum 
is  measured  with  an  accurate  i.o  c.c.  pipette  into  a  rather  wide  test-tube  (about 
18  mm.).  To  the  tube  0.5  to  0.75  c.c.  of  chloroform  is  added  and  the  tube  is 
sharply  shaken,  at  intervals,  until  a  milky  emulsion  is  formed.  We  prefer  chloro- 
form because  the  emulsion  is  more  stable  than  with  ether  or  other  lipoid  solvents. 
A  control  tube  is  inactivated  at  60°  C.  for  thirty  minutes  and  a  drop  of  toluol  is 
then  added  in  place  of  chloroform.  Both  tubes  are  then  incubated  over  night 
(fifteen  to  sixteen  hours  at  37°).  In  the  morning  about  i.o  c.c.  of  a  mixture 
of  10  per  cent,  acetic  acid  plus  20  per  cent,  salt  solution  is  added,  and  the  tubes 
are  then  gently  warmed  in  a  water  bath  until  the  chloroform  has  been  evaporated. 
About  2  or  3  c.c.  of  distilled  water  are  then  added  slowly  and  the  tubes  boiled 
for  at  least  ten  minutes.  The  coagulated  protein  is  filtered  off  by  means  of 
hard  filter  paper,  previously  moistened,  the  filtrate  being  permitted  to  filter 
directly  into  the  large  tubes  used  for  oxidizing.  The  tubes  are  then  oxidized 
and  Nesslerized  according  to  the  usual  Folin  method,  the  readings  being  made 
against  varying  dilutions  of  the  I  mg.  standard,  so  that  test  readings  are  made 
against  standard  of  apparently  equal  color." 

"Serum  esterase  has  been  determined  as  follows:  To  i.o  c.c.  of  the  serum, 
i.o  c.c.  of  neutral,  redistilled  'ethyl  butyrate  and  0.5  c.c.  toluol  are  added,  the 
volume  being  brought  to  10.0  c.c.  with  physiological  salt  solution.  The  flasks 
are  then  shaken  100  times  and  incubated  for  four  hours ;  25  c.c.  of  neutral  95  per 
cent,  alcohol  are  then  added  to  each  flask  and  the  acidity  which  has  developed  is 
titrated  with  N/SO  sodium  hydrate  (alcoholic)  to  a  faint  pink  with  phenolphtha- 
lein.  After  deducting  the  proper  controls,  i.e.,  serum  alone,  ethyl  butyrate  alone, 
etc.,  the  esterase  index  is  expressed  in  terms  of  c.c.  of  N/ioo  sodium  hydrate  used  to 
neutralize  the  acidity  developed  by  i.o  c.c.  of  serum  from  i.o  c.c.  of  ethyl  butyrate." 

Ferment-Antiferment  Balance. — The  activity  of  various  ferments 
in  the  body  is  probably  effective  in  various  degrees  at  all  times,  and 
this  activity  probably  plays  a  certain  part  in  normal  metabolism.  Cer- 


248  THE  PRINCIPLES  OF  IMMUNOLOGY 

tain  of  the  ferments  become  active  only  when  suitable  material  is  pre- 
sented, as  is  the  case  with  the  serum  peptidase;  others  operate  only 
when  the  .surrounding  medium  reaches  suitable  reaction;  still  others 
operate  only  if  the  antiferment  content  is  sufficiently  reduced.  There- 
fore, the  preservation  of  the  body  tissues  against  destructive  action  of 
'ferments  and  the  normal  processes  of  metabolism  depend  in  considerable 
part  upon  the  neutralizing  activity  of  antif  erments. 

Antiferment. — Certain  investigators  have  reported  the  production 
of  specific  antibodies  following  the  injection  of  ferments.  Morgen- 
roth  claimed  to  have  produced  a  specific  antirennin,  Sachs  and  Achalme 
an  antipepsin  and  an  antitrypsin,  Schultze  an  antisteapsin  and  an 
antilactase,  Gessard  an  antityrosinase  and  Moll  an  antiurease.  The 
recent  studies  of  antif  erments,  however,  indicate  that  inhibitory  activity 
is  not  specific  and  this  subject  has  been  contributed  to  particularly  by 
Jobling  and  his  collaborators.  They  are  of  the  opinion  that  anti- 
i erment  activity  depends  upon  the  highly  dispersed  unsaturated  lipoids 
of  the  serum  and  lymph  and  that  the  titer  varies  with  the  amount  of 
lipoids,  their  dispersion  and  chemical  structure.  In  studying  anti- 
trypsin, they  found  that  the  inhibitory  substances  are  of  the  nature 
of  soaps  and  that  the  ability  to  inhibit  ferment  activity  depends  upon  the 
degree  of  unsaturation  of  the  carbon  bonds  in  the  fatty  acid.  They 
made  soaps  from  olive  oil,  cod-liver  oil,  linseed  and  other  oils  and 
found  that  these  soaps  inhibited  the  action  of  trypsin  and  leucoprotease. 
They  determined  further  that  extraction  of  the  blood  serum  with  such 
fat  solvents  as  chloroform  and  ether  removes  the  antitryptic  activity. 
Soaps  prepared  from  the  extracts  restored  the  antitryptic  activity.  The 
serum  residue,  after  extraction,  was  found  to  be  highly  toxic  for 
guinea-pigs.  If,  however,  the  soap  prepared  from  the  extract  were 
added  to  the  residue,  the  toxicity  was  neutralized.  Jobling  and  his 
collaborators  attributed  the  toxic  action  of  the  serum  residue  to  (a) 
alteration  of  the  mechanism  of  intravascular  coagulation,  (&)  exposure 
of  native  serum  proteins  to  the  action  of  ferments  and  (c)  the  resulting 
formation  of  toxic  split  products.  These  workers  isolated  unsaturated 
fatty  acids  from  tubercle  bacilli  and  found  that  when  these  were  sapon- 
ified they  inhibited  the  action  of  trypsin  but  lost  this  power  when  satur- 
ated with  iodine.  They  were  able  to  obtain  similar  soaps  from  caseous 
lymph-nodes  and  suggest  that  the  soaps  prevent  the  activity  of  ferments 
which  would  normally  digest  the  necrotic  material.  This  failure  of 
digestion  leads  to  the  formation  of  the  partly-digested  and  fatty  sub- 
stance which  is  spoken  of  as  the  caseation  necrosis. 

The  antif  erments  are  greatly  augmented  in  certain  diseases,  such  as 
acute  infections,  carcinoma,  cachexias  in  general,  anaphylactic  shock, 
certain  degenerative  changes  of  the  nervous  system  and  in  pregnancy. 
Jobling  explains  the  crisis  of  pneumonia  as  being  due  to  an  alteration 
in  the  ferment-antif  erment  balance ;  that  there  is  a  decrease  in  the  anti- 
ferment  with  a  corresponding  mobilization  of  protease,  an  increase  in 
the  serum  lipase  with  a  resulting  decrease  in  the  non-coagulable  nitrogen 
and  proteoses  of  the  serum. 


DEFENSIVE  FERMENTS  249 

Determination  of  Antiferment  in  Blood  Serum. — The  determination  of 
antitrypsin  by  the  Fuld-Gross  method  is  satisfactory  for  this  purpose.  This 
requires  in  addition  to  blood  serum  taken  preferably  in  the  morning  before 
the  patient's  breakfast,  solutions  of  casein,  acetic  acid,  and  trypsin.  The 
solution  of  casein  is  made  by  dissolving  i  gram  casein  in  100  c.c.  N/io  NaOH 
with  the  aid  of  slight  heating;  the  solution  is  neutralized  with  N/io  NaCl  and 
made  up  to  500  c.c.  with  0.85  per  cent.  NaCl.  The  acetic  acid  solution  is  made 
by  mixing  5.0  c.c.  acetic  acid  with  45.0  c.c.  alcohol  and  50.0  c.c.  water.  The 
trypsin  solution  is  made  by  dissolving  0.5  gram  trypsin  (Griibler)  in  50.0  c.c. 
0.85  per  cent.  NaCl  and  0.5  c.c.  normal  soda  solution;  this  is  diluted  ten  times 
with  saline.  The  patient's  serum  must  be  fresh  and  should  be  diluted  with 
salt  solution  so  as  to  make  a  2  per  cent,  solution. 

The  trypsin  is  titrated  as  follows:  Place  in  a  series  of  test  tubes  o.i,  0.2, 
0.4,  0.6,  0.8,  and  i.o  c.c.  of  the  trypsin  solution.  Add  2.0  c.c.  of  casein  solution 
to  each  tube,  shake  and  incubate  for  one-half  hour  at  50°  C.  Add  three  or 
four  drops  of  the  acetic  acid  solution  to  each  tube  and  note  the  precipitation 
(cloudiness)  which  appears  in  the  course  of  a  few  minutes.  The  tube  which 
remains  perfectly  clear  contains  enough  trypsin  to  digest  2.0  c.c.  of  the  casein 
solution.  For  testing  the  antitryptic  content  of  the  serum  add  0.5  c.c.  of  the 
2  per  cent,  solution  of  serum  to  each  of  six  small  test  tubes.  Then  add  to 
each  tube  in  series,  increasing  amounts  of  the  trypsin  solution,  beginning  with 
the  largest  dose  that  completely  digested  the  casein,  and  increasing  in  each 
tube  by  o.i  c.c.  Add  2.0  c.c.  of  casein  solution  to  each  tube  and  make  up  to 
equal  volumes  with  normal  saline.  Shake  and  incubate  for  one-half  hour  at 
50°  C.;  then  add  three  or  four  drops  of  the  acetic  acid  solution  to  each  tube 
and  observe  as  before.  The  amount  of  trypsin  which  is  inhibited  by  the 
serum  is  determined  by  the  lack  of  complete  digestion,  as  shown  by  the  acetic 
acid  precipitation.  A  control  series  should  be  set  up  with  the  pooled  sera  of 
normal  individuals.  Jobling  and  his  associates  made  the  test  somewhat  more 
accurate  by  filtering  after  the  incubation  and  then  determining  quantitatively 
the  non-coagulable  protein  nitrogen. 

The  Abderhalden  Test. — In  the  discussion  of  the  specificity  of  fer- 
ments, we  pointed  out  that  Abderhalden  had  assumed  that  the  entry  of 
cellular  and  other  proteins  in  the  circulation  could  lead  to  the  formation, 
or  increase,  of  ferments  which  have  as  their  specific  character  the  prop- 
erty of  digesting  the  antigenic  protein.  The  test  is  based  fundamentally 
upon  a  mixture  of  serum  and  antigenic  substance  and  the  determina- 
tion of  the  formation  of  diffusible  protein  products.  He  regarded  the 
ferments  as  protective,  inasmuch  as  they  could  break  down  and  aid 
in  the  elimination  o>f  substances  essentially  foreign  in  nature.  The 
technic  of  the  test  has  been  carefully  reviewed  by  Bronfenbrenner  in 
Vol.  I  of  the  Journal  of  Laboratory  and  Clinical  Medicine,  and  we  call 
particular  attention  to  certain  modifications  that  have  been  offered  by 
Retinger  in  Volume  XXII  of  the  Archives  of  Internal  Medicine.  The 
following  brief  description  of  the  test  is  given  in  order  to  provide  an 
outline  of  the  general  principles. 

The  materials  essential  for  the  test  are  the  serum  or  plasma  of  the 
patient,  the  substratum,  dialyzing  tubes  and  flasks,  carefully  distilled  water, 
clean  test  tubes  and  ninhydrin.  The  blood  is  withdrawn  before  the  patient's 
Ibreakfast  in  order  to  obtain  blood  at  a  time  when  no  dialyzable  products  of 
intestinal  digestion  are  present.  It  may  be  taken  into  paraffin-coated  centri- 
fuge tubes  for  the  preparation  of  plasma  or  may  be  allowed  to  clot  and 
the  serum  centrifuged  so  as  to  be  absolutely  clear.  The  substratum  is  the 
material  to  be  digested.  As  a  rule,  the  tissue  is  cleared  of  connective  tissue 
in  so  far  as  possible,  is  perfused  with  salt  solution  and  subsequently  washed 
several  times  with  distilled  water  until  it  is  absolutely  free  from  blood.  It  is 
then  placed  in  a  suitable  container,  coagulated  by  boiling,  and  repeatedly 
washed  with  boiling  water  until  the  fluid  gives  no  ninhydrin  test.  It  is  then 


250  THE  PRINCIPLES  OF  IMMUNOLOGY 

preserved  under  toluol.  The  dialyzing  thimbles  are  especially  prepared  for 
work  of  this  kind.  They  are  kept  in  distilled  water  for  at  least  a  week  and 
are  carefully  tested  before  use  so  as  to  be  sure  that  protein  does  not  pass 
through  and  also  to  be  sure  that  pepton  will  pass  through.  For  the  actual 
test  a  dialyzing  thimble  is  placed  in  a  clean,  dry,  sterile  Erlenmeyer  flask. 
About  0.5  gram  of  dried  substratum  is  placed  in  the  bottom  of  the  thimble 
and  1.5  c.c.  of  serum  then  introduced.  The  thimble  is  withdrawn,  closed  at 
the  top  by  means  of  a  forceps  and  the  outside  washed  carefully  with  sterile 
water  so  as  to  remove  any  adherent  protein.  The  thimble  is  then  replaced  in 
a  flask  containing  about  20  c.c.  sterile  distilled  water.  The  contents  of  the 
thimble  and  the  water  in  the  flask  are  covered  with  toluol  and  the  flask 
incubated  for  sixteen  to  eighteen  hours.  The  dialyzate  is  examined  by 
means  of  the  nmhydrin  test.  For  this  purpose,  0.2  c.c.  of  i  per  cent,  ninhydrin 
solution  is  placed  in  a  clean  dry  test  tube  and  10.0  c.c.  of  the  dialyzate  added, 
the  mixture  boiled  for  one  minute  and  the  color  observed.  The  development 
of  a  blue  or  violet  color  indicates  the  presence  of  diffusible  protein  products 
and  constitutes  a  positive  test.  Proper  controls  of  all  the  reagents  are  essential. 

Since  the  earlier  work  of  Abderhalden  appeared,  numerous  articles 
have  been  written  and  there  has  been  much  discussion  concerning  the 
alleged  specificity  of  the  reaction.  The  protective  ferments  of  Abderhal- 
den are  assumed  to  possess  the  property  of  directly  digesting  the  antigen 
and  the  appearance  of  the  products  of  such  direct  digestion  constitutes 
the  fundamental  principle  of  the  Abderhalden  test.  Stephan,  Haupt- 
mann,  Bronfenbrenner  and  others  have  shown  that  these  ferments  lose 
their  activity  after  heating  to  58°  C.  for  one-half  hour,  but  they  can 
be  reactivated  by  the  addition  of  fresh  serum.  This  suggests  a  parallel 
with  the  activity  of  complement  and  amboceptor,  but  Frank  and  Rosen- 
thai  point  out  that  in  hemolysis  there  is  no  indication  that  the  action 
of  complement  is  accompanied  by  proteolysis.  Therefore,  although  the 
ferment  may  be  reactivated  after  heating,  this  does  not  necessarily 
indicate  that  it  is  of  the  nature  of  an  amboceptor  or  other  immune 
body.  Flatow,  Plaut  and  others  have  reported  that  positive  results 
can  be  obtained  by  the  manipulation  of  material  and  that  positive  or 
negative  reactions  can  thus  be  found  with  almost  any  serum.  De  Waele 
found  that  he  could  demonstrate  a  digesting  substance  within  a  few 
minutes  after  the  parenteral  introduction  of  foreign  protein,  a  time 
interval  too  short  for  the  production  of  specific  ferments.  Heilner  and 
Petri  regard  this,  however,  as  a  sort  of  mobilization  of  ferment  and  not 
the  result  of  new  formation.  Bronfenbrenner  found  that  the  serum  of 
highly  immunized  animals*  failed  to  digest  the  protein  used  for  im- 
munization. He  determined,  however,  that  such  a  serum  gave  a  positive 
Abderhalden  test  and  with  his  collaborators  has  demonstrated  that  the 
dialyzable  substances  do  not  originate  from  the  substratum.  He  showed 
also  that  the  ferments  responsible  for  the  cleavage  of  protein  during 
the  reaction  are  not  specific.  Positive  results  with  placenta  are  to  be 
obtained  with  the  serum  of  males  as  well  as  of  females,  but  the  protein 
digested  is  that  of  the  serum.  The  work  of  Jobling  and  his  collabor- 
ators favors  the  view  that  proteolytic  activity  of  the  serum  is  not 
specific.  Plaut,  Bronfenbrenner  and  others  found  that  positive 
Abderhalden  tests  may  be  obtained  by  the  use  of  kaolin,  starch,  barium 
sulphate  and  chloroform,  all  of  which  probably  absorb  the  inhibiting 
substance  or  anti ferment  of  the  blood.  Van  Slyke  and  his  associates, 


DEFENSIVE  FERMENTS  251 

by  means  of  determining  the  amino-nitrogen,  found  that  practically  every 
serum  shows  some  degree  of  protein  digestion  when  incubated  with 
placental  tissue.  Van  Slyke's  methods  are  so  accurate  that  it  seems 
probable  that  the  ninhydrin  tests  with  dialyzates  must  vary  consider- 
ably, depending  upon  the  amount  of  dialyzable  substance  which  may 
pass  through  any  given  thimble.  Elsesser  worked  with  the  purified 
vegetable  proteins  of  Osborn  and  found  that  at  best  the  specificity  of 
the  reaction  is  less  than  that  of  anaphylaxis  and  that  there  are  many 
non-specific  results.  Boldyreff  found  that  the  ferments  act  not  only 
upon  placental  proteins  but  also  upon  other  varieties  of  protein;  he 
believes  that  the  method  is  excellent  for  detection  of  proteolytic  enzymes 
in  the  blood  but  as  a  distinctive  sign  of  pregnancy  it  is  useless.  Against 
these  views  are  the  recent  results  of  Retinger,  who  claims  that  it  is 
not  only  possible  to  demonstrate  lesions  of  the  brain  by  this  test  but 
further  to  define  within  fairly  small  limits  the  localization  of  the 
lesion.  It  may  be  that  with  further  modifications  a  test  of  some  clini- 
cal value  can  be  developed  upon  the  basis  of  the  Abderhalden  test.  At 
the  present  time,  there  is  little  reason  for  accepting  the  conception  of 
specific  ferments  and  the  test  has  been  entirely  discarded  in 
many  laboratories. 


APPENDIX  A 
THERAPEUTIC  EMPLOYMENT  OF  BLOOD  SERUM 

INTRODUCTION. 

SERA   PREPARED  BY   USE  OF  BACTERIA  OR  BACTERIAL  EXTRACTS. 

ANTI-STREPTOCOCCUS   SERUM. 

ANTI-MENINGOCOCCUS   SERUM. 

ANTI-PNEUMOCOCCUS    SERUM. 

ANTI-CHOLERA  SERUM. 

ANTI-ANTHRAX   SERUM. 

ANTI-PLAGUE    SERUM. 

ANTI-BACTERIAL  SERUM   FOR  DIPHTHERIA  CARRIERS. 

ANTI-GONOCOCCUS    SERUM. 

ANTI-TUBERCULOSIS   SERUM. 

ANTI-TYPHOID    SERUM. 
AUTO-SERUM  THERAPY. 

GENERAL  USES. 

SYPHILIS. 

HUMAN  IMMUNE    SERUM. 
SERUM  THERAPY  IN  INFECTIONS  OF  UNDETERMINED  ETIOLOGY. 

INTRODUCTION. 

ANTI-POLIOMYELITIS    SERUM. 
ANTI- HOG-CHOLERA  SERUM. 
THERAPEUTIC  USE  OF  NORMAL  SERUM. 

THE  development  of  immunology  has  resulted  in  extensive  study 
of  the  treatment  of  disease  by  sera  prepared  according  to  a  variety  of 
methods.  We  have  considered  in  other  chapters  the  value  of  certain 
sera,  more  particularly  those  which  possess  a  demonstrable  content  of 
antitoxin.  In  this  chapter  there  is  presented  a  brief  statement  as  to  the 
methods  oi  preparation  and  use  of  other  types  of  sera  with  the  idea 
of  illustrating  how  widely  this  form  of  therapeusis  has  extended  and 
the  principles  upon  which  the  methods  are  founded.  Certain  of  these 
sera  have  given  excellent  results,  but  others  have  failed  utterly  and  still 
others  are  yet  in  the  stage  of  experiment  and  investigation.  The  judg- 
ment as  to  the  value  of  many  of  the  sera  rests  upon  statistical  evidence 
collected  on  a  clinical  basis.  The  use  of  man  for  investigation  intrudes 
into  the  results  obtained  a  wide  variety  of  sources  of  error,  many  of 
which  can  be  excluded  in  investigations  upon  the  lower  animals.  Dif- 
ferences in  hygienic  surroundings,  conditions  of  exposure,  presence  of 
diseases  other  than  that  treated,  differences  in  weight,  age  and  sex 
must  all  be  considered.  The  stage  of  increase  or  decrease  of  the  epi- 
demic must  be  included  in  the  final  judgment  since  the  virulence  of 
infections  is  likely  to  be  greater  at  the  beginning  of  an  epidemic  than 
during  its  decline ;  this  may  be  due  to  exhaustion  of  the  causative  agent, 
but  is  more  probably  accounted  for  in  that  the  less  resistant  individuals 
succumb  early  in  the  epidemics  and  the  more  resistant  are  attacked  sub- 
sequently. The  factor  of  error  in  random  sampling  must  be  calculated 
as  closely  as  possible  and  it  must  be  recognized  that  the  greater  the 
number  of  cases  studied,  the  more  conclusive  are  the  results.  The 
252 


EMPLOYMENT  OF  BLOOD  SERUM  253 

investigator  is  always  actuated  by  the  hope  that  the  particular  method 
he  fosters  will  be  of  value  in  the  alleviation  of  human  disease,  and 
this  fact  may  determine  a  subconscious  selection  of  cases  and  perhaps, 
equally  subconscious,  somewhat  superior  nursing  and  better  care  of  the 
cases  under  special  treatment  than  of  the  controls.  Thus  the  analysis  of 
statistical  evidence  must  be  made  with  rigid  consideration  of  the  various 
factors  of  error.  Minor  differences  in  percentages  of  cure  or  of  im- 
provement may  be  within  carefully  computed  factors  of  error  and  still 
not  take  sufficiently  into  account  a  considerable  factor  of  error  resulting 
from  our  ignorance  of  the  intricacy  of  biological  phenomena. 

Immune  sera  for  therapeutic  purposes  have  been  prepared  by  the 
injection  of  bacteria,  their  toxic  or  non-toxic  extracts,  or  by  combina- 
tions of  these  substances.  Some  of  these  sera  exhibit  a  variable  content 
of  antibodies  of  the  first,  second  or  third  order  of  Ehrlich.  The  most 
important  laboratory  test,  however,  appears  to  be  the  protective  value 
of  the  sera  in  preventing*  infection  in  animals  or  their  curative  value 
after  the  infection  is  established.  There  is  no  necessary  parallel  between 
the  content  of  special  antibodies  and  the  protective  or  curative  value,  ex- 
cept in  the  case  of  antitoxic  sera.  Thus  a  serum  may  exhibit  a  low  ag- 
glutinin  or  bacteriolysin  content  and  yet  protect  animals  when  used  in 
extremely  small  amounts.  The  converse  is  also  true,  namely,  that  rela- 
tively high  content  of  agglutinin  or  bacteriolysin  does  not  necessarily 
presuppose  a  great  capacity  for  protection.  Furthermore,  it  cannot  be 
assumed  positively  that  because  animals  are  protected  or  cured,  the 
serum  will  be  of  equal  value  in  human  medicine ;  hence  the  necessity 
for  carefully  studied  experiments  on  man. 

Not  only  have  immune  sera  been  employed,  but  many  attempts  at 
treatment  of  disease  by  means  of  normal  sera  have  been  made.  This 
procedure  is  based  in  part  upon  the  principles  of  non-specific  immun- 
ological  treatment  which  have  been  previously  discussed.  Such  sera 
may  be  obtained  from  man,  horse,  goat,  ox  or  other  animal.  In  the 
treatment  of  certain  hemorrhagic  disease  the  purpose  of  the  serum 
may  be  physiological  rather  than  immunological,  inasmuch  as  the  serum 
is  believed  to  provide  certain  essentials  for  the  process  of  clotting  which 
the  patient  provides  in  insufficient  amounts  or  not  at  all. 

In  the  following  discussion  it  will  be  noted  that  there  are  first  taken 
up  those  sera  prepared  by  immunization  with  bacteria;  second,  those 
prepared  by  immunization  with  bacterial  extracts,  either  with  or  without 
the  bacterial  bodies;  third,  treatment  with  the  patient's  own  serum; 
fourth,  treatment  with  sera  from  convalescent  human  cases;  fifth,  spe- 
cific serum  therapy  in  diseases  of  unknown  origin,  and  finally,  treat- 
ment by  normal  sera. 

SERA  PREPARED  BY  USE  OF  BACTERIA  OR  BACTERIAL  EXTRACTS 

Anti-streptococcus  Serum. — The  protection  afforded  by  the  use  of 
streptococcus  immune  serum  is  still  problematical.  The  reason  for  this 
lies  partly  in  the  fact  that  there  are  several  different  types  of  streptococci 
concerned  in  human  infection.  Some  of  the  strains  occur  frequently  and 


254  THE  PRINCIPLES  OF  IMMUNOLOGY 

others  only  rarely.  It  is,  therefore,  advisable  to  determine  as  soon  as 
possible  the  type  of  the  organism,  and  then  combat  it  with  its  special 
antiserum.  Havens  succeeded  in  dividing  these  organisms  into  three 
distinct  groups  by  means  of  cultural  and  immunological  examination 
and  found  that  an  immune  serum  can  be  produced  for  each  of  the  three 
groups.  The  serum  is  specific  for  its  own  group  and  protects  mice 
against  infection  with  homologous  organisms,  but  furnishes  no  pro- 
tection against  infection  with  organisms  from  the  other  groups.  From 
this  work  it  is  evident  that  the  utilization  of  specific  sera  is  of  para- 
mount importance  in  the  treatment  of  streptococcus  infections.  The 
oldest  serum  is  that  of  Marmorek.  This  serum  was  produced  by  im- 
munization with  a  strain  which  was  made  highly  virulent  by  animal 
passage  and  the  serum  was  found  to  be  protective  experimentally  when 
administered  twelve  to  eighteen  hours  before  the  bacteria  were  injected. 
This  serum  was  used  in  erysipelas,  puerperal  septicemia  and  scarlatinal 
angina  with  favorable  results.  Lenhartz,  Baginsky,  Zangemeister  and 
others,  however,  failed  to  obtain  definitely  good  results  with  anti-strepto- 
coccus sera.  Sera  were  later  produced  by  Aronson  and  Tavel,  Van  de 
Velde,  Meyer,  Ruppel,  Menzer  and  Moser  for  use  in  puerperal  sepsis, 
scarlatina,  erysipelas  and  acute  articular  rheumatism.  In  puerperal 
infection  a  fresh  polyvalent  anti-streptococcus  serum  should  be  given 
daily  in  intravenous  doses  of  30.  c.c.  until  marked  improvement  occurs. 
These  cases  are  usually  slow  in  improvement,  but  the  results  so  far 
obtained  seem  to  be  encouraging.  It  is,  however,  of  the  greatest  im- 
portance to  introduce  serum  treatment  at  the  earliest  possible  moment. 
In  scarlatina  Escherich  found  that  if  the  serum  be  used  on  the  first  and 
second  days  of  illness  recovery  of  the  majority  of  cases  is  likely  to 
occur.  Axenow,  in  fact,  believes  that  it  is  the  only  means  to  ward  off 
a  fatal  outcome.  In  erysipelas  and  acute  articular  rheumatism  the 
results  have  been  at  variance.  Park  states  that  the  injections  should 
be  made  before  the  infection  has  become  advanced  and  before  the 
streptococci  have  acquired  an  increased  resistance  to  the  serum  anti- 
bodies and  ferments.  The  repeated  local  bathing  of  exposed  infected 
tissues  with  the  serum  seems  to  have  a  beneficial  result  beyond  that 
exercised  by  a  non-specific  serum.  The  action  of  anti-streptococcal  sera 
is  largely  due  to  its  opsonic  powers.  The  hope  for  effective  serum 
therapy  in  streptococcus  infection  is  at  present  based  on  the  new 
methods  of  serologic  classification  of  the  organism  and  further  labora- 
tory and  clinical  study  is  highly  desired. 

Anti-meningococcus  Serum. — In  1906  Jochmann  for  the  first  time 
treated  cerebrospinal  fever  with  serum  of  horses  immunized  to  several 
strains,  of  meningococci.  This  serum  was  highly  agglutinative,  some- 
what bactericidal,  but  not  antitoxic.  The  death  rate  among  the  treated 
cases  was  27  per  cent,  as  compared  to  53  per  cent,  among  the  non-treated 
cases.  In  the  earlier  work  Jochmann  administered  the  serum  subcu- 
taneously  but  later  advised  its  use  by  the  intraspinal  method.  Almost 
simultaneously  Flexner  and  Jobling  carried  out  extensive  work  on 
monkeys.  They  demonstrated  that  the  most  beneficial  effect  of  the 


EMPLOYMENT  OF  BLOOD  SERUM  255 

serum  follows  intrathecal  administration,  and  in  1907  successfully 
applied  serum  treatment  of  the  disease  during  an  epidemic  in  Akron, 
Ohio.  The  following  table,  taken  from  Worster,  Drought  and  Mills 
Kennedy,  "  Cerebrospinal  Fever,"  London,  1919,  gives  the  results  ob- 
tained by  several  investigators. 


Author 

Flexner  .  .  . 
Netter  
Dunn 

No. 
of  treated 
cases 

1294  (collected) 

100 
40 

Serum 
used             ' 

Flexner 
Flexner 
Flexner 

Serum 
treated 
mortality     s 

30.9% 
28.0% 

22.5% 

Cases  not 
treated  with 
•erum  mortality 

70% 
49% 

70% 

Robb 

•!&• 
3OO  =*= 

Flexner 

•Ml*^  /L/ 

30.0% 

/  V7  /U 

72% 

Dopter  .  .  . 

Lew 

4O2 

1  6s 

Dopter 
/  Kolle  and      \ 

\IT  r                                    r 

16.4% 

18.8% 

65% 

C2% 

Steiner  .  .  . 
Schoene  .  . 

•*•  WO 

2280  (collected) 
30 

.  Wassermann  J 
Jochmann 

37-0% 
30.0% 

O     f** 

77% 
53% 

Although  many  English  investigators  have  been  successful  in  the  use 
of  anti-meningococcus  serum,  several  experienced  men  have  advised 
against  its  use.  This  is  largely  because  of 'the  fact  that  Gordon,  Ellis 
and  others  have  demonstrated  several  types  or  groups  of  the  meningo- 
coccus,  and  it  is  believed  that  sera  should  be  prepared  against  each 
type  in  order  to  obtain  the  best  results.  Rolleston  has  compiled  the 
following  table  of  results  with  various  types  of  anti-meningococcus  sera : 

Brand  of  serum  Mortality  Recoveries 

Flexner 22.3  per  cent.  77.7  per  cent. 

Gordon  18.7  per  cent.  81.3  per  cent. 

Pasteur  Institute   44.5  per  cent.  55.5  per  cent. 

Burroughs-Wellcome  ...  33.3  per  cent.  66.7  per  cent. 

Mulford   50.0  per  cent.  50.0  per  cent. 

Lister  Institute 54.5  per  cent.  45.5  per  cent. 

Gordon's  and  Flexner's  sera  have  so  far  given  the  best  results.  It 
is  advisable  to  test  the  agglutinative  power  of  a  serum  prior  to  its  use, 
using  a  strain  freshly  isolated  during  the  epidemic.  The  serum  should 
agglutinate  the  organism  in  a  dilution  of  at  least  one  to  five  hundred. 
The  lack  of  a  definite  potency  standard  makes  it  impossible  to  judge 
accurately  the  value  of  any  given  serum.  As  in  diphtheria  and  other 
diseases,  the  early  use  of  serum  is  of  the  greatest  importance.  Flexner 
found  that  if  the  serum  was  given  in  the  first  three  days  the  mortality 
was  1 8  per  cent.,  if  given  from  the  fourth  to  the  seventh  day  it  was 
27  per  cent.,  and  if  given  later  36.5  per  cent.  Similar  figures  were 
obtained  by  Rolleston,  Gray,  Robb  and  Worster-Drought.  If  neces- 
sary, the  injections  should  be  repeated.  According  to  Park,  it  is  advis- 
able to  give  not  less  than  four  daily  injections  unless  the  case  is  already 
convalescent  when  it  comes  under  observation.  If  the  organisms  or 
symptoms  do  not  disappear,  the  injections  of  10  c.c.  to  25  c.c.  of  serum 
should  be  continued  for  many  days.  Finally,  as  a  result  of  army  experi- 
ence, Herrick  believes  that  the  disease  is  in  most  if  not  all  cases  a 
general  bloodstream  infection  with  secondary  meningeal  involvement 
and  therefore  advises  the  use  of  large  doses  of  anti-meningococcus 
serum  intravenously  as  soon  as  the  diagnosis  is  made  in  addition  to 


256 


THE  PRINCIPLES  OF  IMMUNOLOGY 


intrathecal  injections.  The  results  so  far  obtained  seem  to  be  better 
than  when  intraspinal  injections  alone  are  used.  Frich  also  recom- 
mends that  all  patients  with  positive  signs  and  symptoms  be  given  both 
intraspinal  and  intravenous  injections  of  serum.  Large  doses  of  serum, 
both  intravenously  and  intraspinally  at  frequent  intervals  apparently 
do  no  harm,  lower  the  mortality,  prevent  serious  complications  and 
shorten  the  period  of  convalescence. 

Anti-pneumococcus  Serum. — Washburn,  Mennes,  Pane  and  numer- 
ous other  early  investigators  attempted  to  produce  anti-pneumococcus 
sera  for  the  treatment  of  man,  but  their  results  were  irregular  and  not 
encouraging.  An  important  advance  in  the  production  of  anti-pneumo- 
coccus serum  was  made  when  Neufeld  and  Handel  in  1909  pointed  out 
that  pneumococci  can  be  divided  into  various  immunological  groups, 
and  that  no  curative  properties  can  be  expected  from  a  given  serum 
unless  it  is  homologous  for  the  type  that  causes  the  infection.  This 
work  has  been  confirmed  and  extended  by  Dochez  and  Gillespie,  Cole, 
Lister  and  many  others.  At  present  we  recognize  four  groups.  Groups 
I  and  II  are  immunologically  distinct  groups,  Group  III  is  that  of 
the  streptococcus  or  pneumococcus  mucosus,  and  Group  IV  a  heter- 
ogeneous group  of  pneumococci  which  cannot  be  classified  under  the 
other  three  groups.  The  following  table,  taken  from  Park  ("The 
Practical  Application  of  Serum  Therapy,"  Transactions  of  the  Con- 
gress of  American  Physicians  and  Surgeons,  1916,  x,  118)  gives  the 
group-incidence  and  mortality : 


Type 

Number 

Percent,  inci- 
dence 

Per  cent,  mor-o 
tality. 

University  of  Penna. 
Hospital,  Richardson 

Cole 

Longcope 

Cole 

Longcope 

Cole 

Longcope 

No. 

Per  cent, 
incidence 

Per  cent, 
mortality 

I 

78 
75 

22 

48 
H 

*d3) 
(ii) 
(7) 

(21) 

33 
32 
9 

20 

6 

*(23) 
(21) 
(14) 
(40) 

25.0 
29.0 

45-o 
12.5 

*(I2.5) 
(72.7) 
(85-7) 
(23.8) 

60 
39 
13 

83 

31 
2O 

6 
43 

30 
25 
50 
12 

II 

III 

IV 

Other  bacteria  .... 

*  Presbyterian  Hospital,  Longcope. 

From  this  table  it  appears  that  about  30  per  cent,  of  the  cases  of  pneu- 
monia and  about  one-third  of  the  total  deaths  from  the  disease  are 
caused  by  Type  I  pneumococci.  In  the  United  States  Cole  claims  that 
75  per  cent,  of  all  pneumonia  cases  are  caused  by  Types  I,  II  and  III,  and 
25  per  cent,  by  Type  IV.  Lister,  in  South  Africa,  finds  Type  IV  very 
common  among  the  negroes  in  the  Rand.  So  far  only  Type  I  and 
Type  II  sera  have  given  encouraging  results.  The  antigenic  value  of 
Type  III  pneumococcus  is  exceedingly  low,  and  that  of  Type  IV  vari- 
able. From  the  more  recent  work  of  Raphael  it  would  appear  that 
sera  produced  against  various  strains  of  pneumococci  are  in  a  sense 
strictly  monovalent  and  also  that  only  virulent  pneumococci  are  suf- 
ficiently antigenic  to  produce  antisera  of  distinct  value. 

The  sera  against  infection  with  Type  I  organisms  have  been  used 
extensively  and  appear  to  have  given  especially  good  results.     The 


EMPLOYMENT  OF  BLOOD  SERUM  257 

Type  II  antiserum,  however,  is  much  less  efficacious  and  thus  far  its 
therapeutic  value  is  questionable.  The  Types  III  and  IV  antisera 
have  no  clinical  value.  Dochez  reports  sixty-five  cases  treated  with 
Type  I  serum,  with  a  mortality  of  6.6  per  cent.,  as  compared  with  a 
mortality  of  25  per  cent,  in  Type  I  cases  not  treated  with  serum. 
Type  II  cases  treated  with  serum  have  a  mortality  of  25  per  cent, 
as  compared  with  61  per  cent,  without  the  use  of  specific  sera.  When 
patients  are  treated  early,  they  do  well,  and  large  doses  of  serum 
should  be  given  as  soon  as  the  type  of  infection  has  been  determined. 
Cole  advises  an  intravenous  injection  of  80  to  100  c.c.  of  serum  diluted 
with  an  equal  amount  of  salt  solution  and  repeated  every  twelve  hours 
until  improvement  occurs.  The  average  total  amount  of  serum  required 
in  the  Hospital  of  the  Rockefeller  Institute  was  about  250  c.c.  The 
injection  of  such  large  doses  of  serum  is  not  entirely  without  possible 
harm  to  the  patient  because  of  reaction  to  the  foreign  protein.  The 
possibility  of  severe  serum  sickness  should  further  be  taken  into  con- 
sideration. From  evidence  recently  collected,  more  particularly  in  the 
United  States  Army,  the  value  of  anti-pneumococcus  sera  has  been 
questioned.  Parallel  series  of  cases  showed  no  important  difference 
in  mortality  between  series  receiving  anti-pneumococcus  serum,  normal 
horse  serum  or  no  serum  whatever.  The  patients  were  young  men  who 
had  passed  rigid  physical  examinations  and  therefore  were  good  risks 
in  acute  infections.  It  does  not  follow  that  other  classes  of  patients 
would  show  the  same  results.  There  is  also  great  variation  in  the  mor- 
tality of  different  epidemics,  and  also  normally  in  different  ages,  so 
that  only  a  sufficiently  large  number  of  treated  cases  extensively  con- 
trolled will  form  a  trustworthy  basis  of  actual  comparison  as  to  the 
death  rate,  which  is,  after  all,  the  final  criterion  as  to  the  actual  value 
of  the  serum.  Cecil  and  Blake  have  recently  examined  the  question 
on  the  basis  of  experiments  with  monkeys.  They  find  that  the  admin- 
istration of  normal  horse  serum  has  no  beneficial  effect  on  experimental 
pneumococcus  Type  I  pneumonia  but  that  the  intravenous  administra- 
tion of  specific  Type  I  antiserum,  particularly  if  given  early  and  fre- 
quently, "  exercises  a  specific  therapeutic  effect,  frees  the  blood 
promptly  and  permanently  from  pneumococci,  shortens  the  course  of 
the  disease  and  greatly  moderates  its  severity."  The  treatment  of 
lobar  pneumonia  with  Cole's  serum  at  present  is  best  carried  out  in 
institutions  where  it  is  possible  to  make  accurate  bacteriological  diag- 
nosis and  differentiation  of  the  types  of  the  cocci,  and  where  intra- 
venous administration  of  large  doses  of  sera  can  be  accomplished  with 
the  largest  margin  of  safety  to  the  patient. 

Kyes  has  carried  out  extensive  investigations  on  the  clinical  value  of 
a  serum  produced  by  injecting  massive  doses  of  virulent  pneumococci 
into  the  domestic  fowl.  The  reason  for  the  selection  of  the  fowl  as 
supply  animal  is  that  no  matter  how  virulent  pneumococci  are  for  other 
species,  they  do  not  occasion  disease  in  fowls,  and  therefore  large  doses 
can  be  injected  with  impunity.  The  initial  dose  in  most  instances  is  a 
surface  growth  equal  to  that  of  240  test-tube  slants.  The  average 
17 


258  THE  PRINCIPLES  OF  IMMUNOLOGY 

subsequent  doses  are  approximately  400  test-tube  slants  each.  All  the 
injections  are  made  intraperitoneally.  Injections  are  given  every  two 
weeks  over  periods  of  from  four  months  to  two  years.  One  week  after 
the  sixth  injection  a  trial  bleeding  is  made  and  thereafter  at  intervals  of 
two  weeks,  alternating  with  the  biweekly  injections.  The  sera  possess 
a  high  content  of  agglutinins  and  bacteriolysins  and  also  exhibit  a 
marked  therapeutic  influence  upon  infected  animals.  Clinically  the 
serum  is  used  in  doses  of  2.5  c.c.,  and  injections  made  slowly.  A  ma- 
jority of  the  cases  have  received  one  injection  daily,  but  not  infre- 
quently two  injections  are  given  the  same  day.  The  injections  are 
continued  until  the  temperature  remains  below  100°  F.  Of  538  cases 
not  treated,  244  cases  died,  the  death  rate  being  45.3  per  cent.  Of  the 
175  similar  cases  treated  with  serum  the  death  rate  was  20.8  per  cent. 
In  the  ward  in  which  the  serum  was  employed  the  death  rate  during 
the  six  weeks  prior  to  the  introduction  of  the  serum  treatment  was  55 
per  cent.  During  the  six  weeks  subsequent  to  the  withdrawal  of  the 
serum  treatment,  the  death  rate  was  51  per  cent.  These  results  are 
distinctly  encouraging.  McClelland  has  recently  reported  the  results 
in  322  cases  of  lobar  pneumonia  in  soldiers  at  Camp  Grant  in  which 
treatment  with  fowl  serum  was  given  and  concludes  that  the  low 
mortality  (7.7  per  cent.)  together  with  the  favorable  modification  of 
clinical  symptoms  by  the  serum  would  seem  to  indicate  the  extension 
of  its  use  in  pneumococcus  pneumonia.  Considering  the  fact  that 
these  cases  were  in  selected  young  men  of  military  age,  and  that  the 
author  does  not  give  a  comparative  mortality  among  non-treated  cases, 
much  of  the  value  of  this  paper  is  lost.  The  serum  has  also  been  used 
by  Litchfield  with  great  benefit  in  a  series  of  pneumococcus  meningitis 
cases.  Gray  employed  the  Kyes  serum  in  234  cases  of  pneumococcus 
pneumonia  with  a  mortality  of  16.8  per  cent.,  whereas  in  similar  cases 
treated  in  the  same  way  except  that  they  received  no  serum,  the  mor- 
tality was  63.6  per  cent.  Much  laboratory  and  clinical  work  remains  to 
be  done  before  any  definite  conclusive  evidence  as  to  the  value  of 
polyvalent  or  antigroup  sera  can  be  drawn  with  any  degree  of  safety. 
Pneumococcus  sera  act  in  part  by  opsotiization  of  the  cocci,  thus  favor- 
ing phagocytosis.  The  standardization  requirements  of  the  Hygiene 
Laboratory,  Washington,  call  for  a  serum  that  shall  protect  white  mice 
against  Type  I  pneumococcus  only.  It  is  felt  by  Ferry  and  Blanchard 
and  many  others  that  a  potent  polyvalent  serum  is  an  absolute  necessity. 
These  authors  recently  succeeded  in  immunizing  horses  with  Types  I, 
II  and  III  and  some  strains  of  Type  IV.  This  serum  in  doses  of  0.2  c.c. 
protected  mice  against  infection  of  Types  I,  III  and  IV  organisms  (ten 
million  M.L.D.). 

Anti-cholera  Sera. — While  antisera  against  cholera  have  been  pro- 
duced by  several  investigators,  the  treatment  of  the  disease  with  these 
sera  has  not  given  the  best  of  results.  Metchnikoff,  Roux  and  others 
have  prepared  sera  against  the  toxins  of  organisms  cultivated  in  col- 
lodion sacs.  McFadyen  used  ground  organisms.  Kraus  used  the  toxin 
of  the  El  Tor  vibrio  as  antigen.  This  organism  was  obtained  by 


EMPLOYMENT  OF  BLOOD  SERUM  259 

Gottschliech  in  1905  from  the  intestinal  contents  of  pilgrims  who  had 
died  at  El  Tor  from  dysentery,  and  is  not  a  true  cholera  vibrio  but 
very  closely  related  to  it.  Kraus  recommended  his  antitoxin  for  the 
treatment  of  the  cholera.  Schurupoff  treated  one-and-a  half  to  two- 
day-old  cultures  of  the  vibrio  with  alkali  and  injected  this  toxic  material 
into  horses  at  six  to  ten-day  intervals.  Under  Kolle's  direction,  Car- 
riere and  Tomarkin  injected  horses  and  goats  with  cholera  cultures  con- 
taining also  the  toxic  derivatives  and  used  the  mixed  sera  of  these 
animals.  They  found  that  these  sera  are  more  valuable  against  cholera 
peritonitis  of  guinea-pigs  than  any  other  animal. 

Ketscher  and  Kernig  used  Kraus'  serum  in  119  severe  and  mod- 
erately severe  cases  with  a  death  rate  of  58  per  cent,  in  those  who 
received  subcutaneous  injections,  and  50  per  cent,  when  used  intrave- 
nously, while  among  the  non-injected  cases  the  mortality  was  63.4  per 
cent.  Others  have  found  among  the  serum-treated  cases  a  mortality  of 
57.5  per  cent,  and  among  the  control  cases  84.3  per  cent.  This  serum 
was  administered  by  Jegunoff  intravenously  together  with  physiological 
salt  solution,  giving  at  first  140  c.c.  of  serum  with  500  c.c.  to  700  c.c.  of 
physiological  salt  solution  and  subsequently  a  second  injection  of  80  to 
1 20  c.c.  of  serum  within  seven  and  one-half  to  twenty-three  hours  after 
the  primary  injection.  During  the  Russian  epidemics  in  1908  and  1909 
it  was  shown  that  large  doses  of  sera  did  not  harm  the  patients.  It 
was  originally  believed  that  large  doses  of  sera  lead  to  quick  destruc- 
tion of  the  vibrios  with  subsequent  intoxication,  but  this  has  not  proven 
to  be  the  case.  During  these  epidemics  Salimbeni's  and  Kraus'  sera 
did  not  give  satisfactory  results.  SchurupofFs  serum  was  considerably 
better,  and  the  best  results  were  obtained  with  the  serum  prepared 
according  to  Carriere  and  Tomarkin.  This  serum  was  given  in  doses 
of  50  c.c.  to  100  c.c.  diluted  with  salt  solution  subcutaneously  and  intra- 
venously and  resulted  in  quick  improvement.  Von  Stiihlern  and 
Tuschinski  treated  149  algid  cases  with  fifty-six  deaths.;  twenty-five 
moderate  and  thirteen  early  cases  were  treated  with  no  deaths.  From 
a  total  of  187  cases  the  mortality  was  29.9  per  cent.  The  serum  should 
be  applied  as  early  as  possible.  Cholera  antisera  contain  bacteriolytic, 
agglutinative,  probably  anti-endotoxic,  complement-fixing  antibodies, 
and  also  tropins.  Because  of  the  variety  of  sera  used  and  the  incon- 
clusive reports  given  it  is  exceedingly  difficult  to  reach  a  definite  con- 
clusion regarding  the  curative  value  of  anti-cholera  sera.  It  seems  to 
us  that  Carriere  and  Tomarkin's  serum  is  the  most  promising. 

The  Use  of  Anti-anthrax  Serum. — The  treatment  of  anthrax  has 
consisted  mainly  in  excision  of  the  pustule,  application  of  chemical  or 
thermal  cautery,  and  the  injection  of  germicides  as  iodine,  mercuric 
chlorid  or  phenol  in  the  regions  of  the  pustule,  but  all  these  methods 
are  objectional  because  they  are  likely  to  produce  scars  and  disfigure- 
ment. Excision  may  furthermore  increase  the  danger  of  systemic 
infection.  Sclavo,  Deutsch,  Sobernheim  and  others  have  produced 
immune  sera  by  immunization  of  the  sheep,  horse  and  ass  with  attenu- 
ated culture  of  anthrax  bacilli.  From  the  work  of  Marchoux  we  know 


26o  THE  PRINCIPLES  OF  IMMUNOLOGY 

that  this  serum  possesses  prophylactic  and  therapeutic  properties  in 
animals.    Anti-anthrax  serum  has  been  used  for  several  years,  espe- 
cially in  Italy,  France  and  England,  with  encouraging  results.     Sclavo 
treated  his  cases  without  excision  and  in  a  series  of  164  cases  treated 
with  specific  serum  this  author  reduced  the  mortality  from  24  per  cent. 
to  5.3  per  cent.     Sclavo  recommends  30  to  40  c.c.  serum  administered 
subcutaneously  in  doses  of  10  c.c.  on  the  first  day  and  repeated  if  neces- 
sary on  the  next  day.    In  severe  infections  10  c.c.  were  given  by  the 
intravenous  route.    In  severe  cases  it  is  advisable  to  give  the  injections 
in  massive  doses  of  80  c.c.  to  100  c.c.  and  preferably  intravenously. 
Shera  advises  administration  of  20  c.c.  every  twelve  hours  until  pyrexia 
ceases.    Regan  recently  injected  the  serum  (10  c.c.  to  15  c.c.)  into  the 
tissues  surrounding  the  pustule  and  found  that  it  possesses  none  of  the 
disadvantages  of  the  previous  methods  of  local  treatment,  and  has  a 
very  rapid  and  complete  effect  on  the  pustule,  not  only  in  arresting  its 
further  development  but  also  in  producing  a  subsidence  of  all  local 
inflammatory  symptoms.     He  advises  also  general  treatment  either 
intramuscularly  or  intravenously.     Local  treatment  in  order  to  effect 
a  cure  must  anticipate  the  onset  of  an  anthrax  septicemia.     In  case 
the  organisms  have  been  demonstrated  in  the  bloodstream  the  prog- 
nosis is  usually  grave.    In  this  case  200  c.c.  of  serum  is  not  excessive, 
and  if  necessary  should  be  repeated  until  a  negative  blood  culture  is 
obtained.    In  intestinal  anthrax  large  doses  of  serum  should  be  given 
by  the  intravenous  route.    Penna  and  Beltrami  and  Penna,  Cuenca  and 
Kraus  have  obtained  good  results  with  normal  beef  serum.    Their  mor- 
tality was  6.2  per  cent,  in  372  cases,  while  the  mortality  previous  to  this 
period  of  treatment  was  about  10  per  cent.    These  authors  advise  the 
use  of  30  to  50  c.c.  of  normal  beef  serum  administered  subcutaneously. 
If  no  improvement  occurs  the  injections  should  be  repeated  every 
twelve,  twenty-four  or  thirty-six  hours,  but  it  seldom  happens  that  a 
patient  requires  more  than  two  or  three  injections.     In  severe  cases 
intravenous  administration  is  recommended.    Similar  favorable  results 
were  obtained  by  Solari  and  Langon,  but  Lignieres  has  reported  un- 
favorably upon  the  curative  action  of  normal  beef  serum,  stating  that 
it  is  inferior  to  horse  anti-anthrax  serum.     He  calls  attention  to  the 
prevalence  of  anthrax  in  cattle  as  evidence  of  the  apparent  lack  of 
natural  resistance  to  the  disease.    More  recently  Kolmer,  Wanner  and 
Koehler  pointed  out  on  the  basis  of  their  experiments  that  normal  beef 
serum  as  secured  from  animals  under  ordinary  conditions  is  but  feebly 
protective  or  curative  for  anthrax  and  while  its  administration  as 
described  by  Penna  and  his  associates  may  favorably  influence  the  pus- 
tule it  is  doubtful  if  the  serum  is  sufficiently  powerful  to  influence 
anthrax  bacteremia.     According  to  Kolmer,  cases  with  sterile  blood 
culture  always  recover.     The  potent   factor  of   anti-anthrax   serum 
appears  to  be  a  thermostable  opsonin. 

The  Serum  Treatment  of  Plague. — The  therapeutic  value  of  anti- 
plague  serum  is  still  a  matter  of  dispute.  Plague  epidemics  are  exceed- 
ingly variable  in  character.  Irregularity  in  the  gravity  of  the  disease 


EMPLOYMENT  OF  BLOOD  SERUM  261 

in  different  individuals  is  of  common  occurrence.  A  wide  variety 
of  antisera  has  been  employed,  but  no  attempt  has  been  made  to  stand- 
ardize the  different  sera.  In  many  instances  the  cases  treated  were 
especially  selected  and  moribund  cases  excluded.  The  result  is  that  much 
of  the  existing  statistical  data  is  unreliable.  Yersin,  Calmette  and  Borrel 
were  the  first  to  show  that  the  serum  of  an  animal  immunized  to  bacillus 
pestis  has  protective  qualities  and  Yersin  is  credited  with  the  production 
of  anti-plague  horse  serum.  This  serum  was  prepared  by  immunization 
of  horses  first  with  dead  and  subsequently  with  living  bacilli.  Tavell's 
serum  was  prepared  on  the  same  principle,  but  Hata  and  also  Kraus 
immunized  their  animals  with  dead  bacilli  alone  and  claim  that  these 
sera  compare  favorably  with  sera  produced  by  the  injection  of  living 
bacilli.  The  use  of  dead  bacilli  minimizes  the  danger  of  laboratory  in- 
fections. Soon  after  the  discovery  of  the  nucleoproteins  by  Ferrannini, 
Galeotti  and  Lustig  employed  nucleoproteins  from  plague  bacilli  as 
antigen  for  the  production  of  anti-plague  serum.  For  this  purpose  the 
bacilli  were  broken  down  in  i  per  cent.  KOH  solution  and  the  nucleo- 
proteins precipitated  by  the  addition  of  acetic  acid  and  then  suspended 
in  salt  solution.  Rowland  also  used  a  similar  antigen,  and  others  have 
employed  a  variety  of  extracts  as  antigens. 

The  serum  at  present  most  commonly  used  is  obtained  from  horses 
after  repeated  intravenous  injections  of  killed  cultures  sometimes  fol- 
lowed by  living  organisms.  Experimentally  the  sera  show  considerable 
strength  in  protecting  animals  against  infection  and  exhibit  specific 
bacteriolytic,  bacteriotropic,  agglutinative  and  antitoxic  qualities.  The 
antitoxic  titer  is  usually  very  low.  According  to  Kraus,  Yersin's  serum 
is  not  any  better  than  the  sera  prepared  with  dead  bacilli  or  nucleo- 
proteins. Yersin  used  his  serum  in  twenty-six  cases  during  the  epi- 
demics of  1896  in  Canton  and  Amoy,  China,  with  a  mortality  of  7.6 
per  cent.,  while  the  mortality  in  cases  not  treated  with  serum  reached 
80  per  cent,  to  90  per  cent.  In  1897  141  cases  were  treated  in  Bombay 
and  Cutch-Mandir  with  a  mortality  of  49  per  cent.  Of  685  cases  not 
treated  80  per  cent.  died.  In  1898  thirty-three  cases  were  treated  in 
Anam  with  Yersin's  serum.  The  death  rate  among  non-treated  cases 
was  100  per  cent,  but  was  42  per  cent,  among  the  serum-treated  cases. 
It  was  found  that  the  serum  was  entirely  inefficient  in  cases  with  the 
pneumonic  form  of  the  disease.  The  German  Commission  at  Bombay 
claimed  that  the  low  mortality  (50  per  cent.)  o>f  serum-treated  cases  was 
due  to  the  selection  of  mild  cases  or  cases  arriving  at  the  hospitals 
during  the  first  or  second  day  of  their  illness.  Clemon  also  failed  to 
obtain  results  in  his  fifty  cases  in  which  he  injected  as  much  as  60  c.c. 
of  the  Yersin  serum.  The  Indian  Plague  Commission  did  not  report 
favorably  on  Yersin's  serum.  Calmette  and  Salimbeni  obtained  very 
good  results  with  serotherapy  in  Oporto,  Portugal ;  of  142  treated  cases 
twenty-one  died,  while  of  seventy-two  not  treated  forty-six  died.  Kos- 
sel  and  Frosch  and  others  studied  this  epidemic  and  found  it  to  be  of 
a  mild  type.  During  the  Manchurian  campaign  serum  treatments 
were  entirely  inefficient.  Choksy  injected  large  doses  (100  c.c) 


262  THE  PRINCIPLES  OF  IMMUNOLOGY 

of  the  Parisian  serum  in  his  cases,  repeating  this  six  to  eight 
hours  later,  and  if  necessary  followed  again  by  another  injec- 
tion. The  next  two  days  he  administered  20  to  50  c.c.,  so  that 
an  adult  received  a  total  of  590  c.c.  From  a  careful  study  he  obtained 
a  mortality  of  72.5  per  cent,  among  the  serum-treated  cases,  and  82.3 
per  cent,  among  his  control  cases.  This  author  also  emphasizes  the 
enormous  advantage  of  early  injections.  In  a  series  of  222  cases 
treated  on  the  second,  third,  fourth,  fifth,  sixth  and  seventh  day  of 
illness  he  found  the  mortality  as  follows:  38.2,  56.7,  58.2,  50.8,  62.9, 
60.0,  and  75  per  cent,  respectively.  According  to  Burnet,  satisfactory 
results  have  been  obtained  in  Queensland  at  the  Colmolie  Plague  Hos- 
pital. Among  190  serum-treated  cases  during  the  period  1901  to  1907 
the  mortality  was  29.7  per  cent.,  while  the  mortality  during  the  same 
period  among  non-treated  cases  was  73.9.  Penna  in  Argentina  injects 
massive  doses  80  to  100  c.c.  intravenously,  and  repeats  the  injection  of 
50  c.c.  after  twelve  to  twenty-four  hours.  Among  664  treated  cases 
during  the  period  1905  to  1912  he  reported  a  mortality  as  high  as  23 
per  cent,  in  1906,  and  a  mortality  of  7.3  per  cent,  in  1912,  the  average 
mortality  was  12.5  per  cent.  From  1914  to  the  middle  of  1919  Kraus' 
serum  was  used  with  an  average  mortality  of  7.8  per  cent.  Kraus' 
serum,  therefore,  gave  better  results  than  Yersin's  serum.  Intravenous 
or  intramuscular  injections  can  be  employed  to  ensure  rapid  absorption 
and  the  injections  should  be  continued  every  twelve  to  twenty-four  hours 
for  two  or  more  days  until  diarrhea  has  been  controlled  and  the  disease 
begins  to  subside.  From  all  these  studies  we  may  conclude  that  although 
serum  therapy  of  plague  has  not  given  striking  results  as  diphtheria 
antitoxin  in  diphtheria,  still  it  is  the  only  specific  means  of  combat- 
ing the  disease  and  when  given  early  and  in  massive  doses  appar- 
ently influences  the  disease  favorably. 

Anti-bacterial  Serum  in  the  Treatment  of  Diphtheria  Carriers. — 
Although  Wassermann  in  1902  recommended  the  use  of  a  bactericidal 
serum,  Martin  was  the  first  to  use  anti-bacterial  serum  in  the  treatment 
of  diphtheria  carriers.  Martin  injected  diphtheria  bacilli  intravenously 
or  intraperitoneally  into  horses  and  obtained  sera  with  marked  agglu- 
tinating properties.  He  claims  that  this  serum  has,  when  applied 
locally,  the  property  of  causing  a  rapid  decrease  in  number  of  living 
bacilli  in  the  throat.  The  best  results  were  obtained  by  incorporating 
the  dried  serum  with  gum  and  using  it  in  the  form  of  pastilles.  Dopter 
and  many  others  have  reported  a  decrease  in  the  carrier  period  by  the 
use  of  anti-bacterial  serum.  More  recently  Roskam  and  Arloing  and 
Stevenin  have  called  attention  to  the  value  of  this  method  of  treatment. 
Ecker  immunized  sheep  with  various  strains  of  diphtheria  bacilli,  and 
by  using  massive  doses  obtained  a  potent  agglutinative  and  lytic  serum. 
To  this  serum  fresh  guinea-pig  complement  was  added  and  the  mixture 
sprayed  by  means  of  atomizers  into  the  nasal  passages,  and  over  tonsils, 
fauces  and  pharynx  four  and  five  times  a  day.  A  total  of  forty-eight  cases 
were  treated,  eighteen  convalescent  and  thirty  contact  carriers.  The 
duration  of  the  carrier  state  after  the  introduction  of  the  serum  was  seven 


EMPLOYMENT  OF  BLOOD  SERUM  263 

days,  while  the  average  duration  of  eighty-seven  control  cases  was  18.6 
days.  A  few  cases  proved  to  be  persistent  carriers.  The  less  favorable 
results  obtained  by  Kretschmer,  Blumenau  and  Nolf  may  be  explained 
by  their  small  series  of  treated  cases,  weak  sera  and  ineffective  methods 
of  application  of  the  serum.  Although  the  results  so  far  obtained  are 
not  entirely  convincing,  the  use  of  anti-bacterial  serum  in  the  treatment 
of  diphtheria  still  deserves  careful  consideration.  It  is  not  pos- 
sible to  resort  to  tonsillectomy  or  adenoidectomy  in  all  instances, 
and  the  majority  of  antiseptics  are  irritant.  In  many  instances  it  is 
practically  impossible  to  reach  the  organisms  because  they  are  buried 
in  crypts,  and  tonsillectomy  remains  as  the  favored  mode  of  treatment, 
although  even  this  method  is  not  invariably  successful. 

Anti-gonococcus  Sera. — The  early  work  of  Rogers  and  Torrey 
has  led  to  attempts  at  treatment  of  gonococcal  infections  by  means  of 
immune  sera.  Torrey's  serum  is  prepared  by  injecting  sheep  with 
dead  and  subsequently  living  cultures  of  virulent  strains  of  the  gono- 
coccus.  Although  efforts  have  been  made  to  treat  urethral,  vulvar 
and  vaginal  gonorrhea  by  local  applications  of  serum  the  disposition 
of  the  organisms  in  deep  glands  has  been  sufficient  to  result  in  the 
failure  of  this  method.  Recent  studies  of  Debre  and  Paraf  offer  some 
encouragement  for  the  treatment  of  gonorrheal  rheumatism  by  the  use 
of  polyvalent  sera,  but  they  find  that  local  injections  about  the  site 
of  the  disease  are  more  effective  than  general  subcutaneous  or  intra- 
venous injections.  Further  studies  may  demonstrate  the  value  of 
serum  treatment  of  chronic  gonorrheal  infections,  but  at  the  present 
time  the  method  cannot  be  highly  recommended. 

Serum  Treatment  of  Tuberculosis. — The  best-known  sera  for  use 
in  tuberculosis  are  those  of  Maragliano  and  Marmorek.  The  first  is 
prepared  by  immunizing  horses  with  a  mixture  of  a  toxic  filtrate  of 
the  bacilli  and  an  aqueous  extract  of  killed  virulent  tubercle  bacilli. 
One  cubic  centimetre  of  the  immune  horse  serum  is  injected  into  the 
patient  every  other  day  for  a  period  of  one  and  one-half  months.  A 
number  of  Italian  workers  found  the  serum  effective,  but  other  ob- 
servers have  not  been  convinced  of  its  value.  Marmorek's  serum  is 
prepared  by  inoculating  horses  with  young  tubercle  bacilli  poor  in  acid 
fast  character.  In  addition,  Marmorek  immunized  animals  with  pure 
cultures  of  streptococci  obtained  from  the  sputum  of  tuberculous 
patients.  This  serum  is  injected  subcutaneously  in  daily  doses  of  from 
5  to  10  c.c.  or  intrarectally  in  doses  of  from  10  to  20  c.c.  A  number  of 
workers,  as  for  instance  Wohlberg,  have  reported  a  favorable  influence ; 
others  deny  this  effect.  Wohlberg  found  the  best  results  in  scrofulous 
cases  but  not  in  cases  of  fully  developed  tuberculosis.  The  benefits  of 
serum  therapy  of  tuberculosis  have  not  been  convincing. 

Serum  Treatment  of  Typhoid  Fever. — Lewin  and  Yes,  Beumer 
and  Pf eiffer  and  Chantemesse  were  among  the  first  to  produce  antisera 
for  this  disease.  Chantemesse's  antiserum  was  prepared  by  immuniz- 
ing horses  with  soluble  toxins  of  the  typhoid  bacillus.  Balthasard 
tested  this  serum  and  found  it  to  agglutinate  typhoid  bacilli  in  very 


264  THE  PRINCIPLES  OF  IMMUNOLOGY 

high  dilutions  and  to  protect  animals  under  experimental  conditions. 
In  1000  cases  of  typhoid  fever,  Chantemesse  reduced  the  mortality  to 
4.3  per  cent,  whereas  the  mortality  among  5621  cases  at  the  other 
hospitals  in  Paris  not  treated  with  serum  was  17  per  cent.  Similar 
favorable  reports  were  made  by  Brunon  and  Josias.  Kraus  and 
Stenitzer  also  produced  antitoxic  sera  by  immunizing  their  animals 
with  soluble  toxins  and  Cjaupp  claims  that  the  serum  can  be  used  with 
advantage  in  the  treatment  of  the  disease.  Besredka  and  Liidke  pre- 
pared sera  by  immunizing  horses  and  goats  with  the  endotoxin  of  the 
typhoid  bacillus,  but  it  seems  that  the  serum  is  not  primarily  an  anti- 
endotoxin  but  rather  a  bactericidal  serum  which  neutralizes  both  the 
exo-  and  endotoxins  of  the  typhoid  bacillus.  According  to  Andriesen 
and  Cinca,  it  can  be  used  clinically.  Sera  were  also  prepared  by  im- 
munizing animals  with  sensitized  cultures  of  the  typhoid  bacillus  and 
also  with  products  obtained  by  digesting  typhoid  bacilli  with  trypsin. 
This  toxic  compound  is  known  as  "  Fermotoxin "  (Gottstein  and 
Mathes).  Rommel  and  Herman  failed  to  obtain  encouraging  results 
with  serum  prepared  by  immunization  with  sensitized  bacilli.  The  most 
favorable  results,  however,  were  secured  by  Rodet  and  Langrifoul. 
These  authors  immunized  horses  intravenously  with  both  living  cultures 
and  old  endotoxins,  and  in  a  summary  of  400  cases  Rodet  finds  that  by 
repeated  injections  of  this  serum  in  doses  from  10  to  20  c.c.  given  sub- 
cutaneously  every  other  day  the  duration  of  the  fever  is  markedly 
reduced  in  cases  that  are  treated  early.  Serum  treatment  appears  to 
reduce  the  bacteremia.  It  is  also  known  that  twenty-four  hours  after 
the  injection  of  serum  a  definite  increase  in  splenic  dullness  is  observed, 
which  presumably  indicates  a  general  stimulation  of  the  lymphoid  and 
myeloid  tissue.  The  self-limitation  of  the  disease,  in  the  absence  of 
complications,  throws  some  doubt  on  the  practical  value  of  such  sera. 

AUTO-SERUM  THERAPY 

The  use  of  the  patient's  own  serum  in  the  treatment  of  his  disease 
has  been  suggested  and  applied  by  a  number  of  workers.  Gilbert, 
Marcon  and  many  others  treated  tuberculous  peritonitis  and  tubercu- 
lous pleurisy  with  effusion,  by  the  subcutaneous  injection  of  I.  c.c.  to 
2.  c.c.  of  the  patient's  own  serum  and  claim  that  the  absorption  of  the 
exudate  is  greatly  increased  and  an  immediate  improvement  occurs. 
Eisner  observed  a  leucocytosis  following  the  injection  of  the  serum 
in  experimental  tuberculous  infections  of  rabbits  and  guinea-pigs  and 
believes  that  this  fact  explains  the  favorable  results  reported  in  this 
method  of  treatment.  Other  investigators  believe  that  specific  antibodies 
favorably  influence  the  process,  but  Levy,  Valenzi  and  others  are 
inclined  to  believe  that  the  results  are  independent  of  the  injections. 
It  is  possible  that  simultaneously  with  the  transfer  of  the  serum  a 
minute  amount  of  tuberculin  is  introduced.  The  exact  nature  of  the 
phenomenon  is,  however,  obscure.  In  influenza,  Malta  fever  and 
typhoid  fever  Modinos  has  also  obtained  beneficial  effects  and  Jez 
applied  the  treatment  favorably  in  erysipelas.  Capogrossi  more  re- 


EMPLOYMENT  OF  BLOOD  SERUM  265 

cently  treated  two  cases  of  cerebrospinal  meningitis  with  fairly  good 
results.  Hodenpyl  treated  a  case  of  carcinoma  with  the  patient's  own 
ascitic  fluid  with  apparent  success.  He  used  this  fluid  in  large  quanti- 
ties in  a  number  of  cases  but  only  with  transient  success.  Risley  also 
applied  this  method  of  treatment  in  sixty-five  cases  of  cancer,  using 
ascitic  fluid  from  cancer  cases  and  also  other  body  fluids  from  non- 
cancerous  cases.  No  encouragement  for  this  method  has  been  found 
in  experimentally  inoculated  mouse  cancers  and  the  subsequent  history 
of  Hodenpyl's  cases  showed  no  permanent  improvement.  Auto-serum 
therapy  has  further  been  applied  in  obstinate  and  chronic  skin  troubles, 
such  as  psoriasis,  dermatitis  herpetiformis,  pemphigus,  lichen  ruber, 
lichen  planus,  urticaria  and  squamous  eczema.  The  serum  is  used  in 
doses  of  30  to  40  c.c.  and  repeated  from  two  to  six  times  at  intervals 
of  from  three  to  five  days. 

Auto-serum  Therapy  in  Syphilis. — Perhaps  the  most  widely 
used  auto-serum  therapy  is  the  salvarsanized  auto-serum  in  the 
treatment  of  parasyphilis.  The  treatment  of  syphilis  of  the  ner- 
vous system  with  salvarsan  or  neosalvarsan  alone  has  not  given 
the  results  expected.  This  is  because  the  choroid  plexus  filters 
out  these  compounds,  preventing  their  entry  into  the  cerebrospinal 
fluid.  It  has  been  shown  by  Plaut  that  the  serum  of  patients  who  have 
received  salvarsan  possesses  antisyphilitic  power,  while  normal  serum 
fails  to  display  this  characteristic.  Similarly  Meirowsky  and  Hart- 
mann  and  Gibbs  and  Calthrop  obtained  good  results  in  the  subcutaneous 
treatment  of  lues  with  serum  of  salvarsanized  patients.  According  to 
Swift  and  Ellis,  salvarsanized  serum  inhibits  the  treponema  more  in- 
tensively if  heated  to  56°  C.  for  half  an  hour.  These  facts  formed  the 
underlying  principles  for  the  treatment  of  late  syphilis  with  salvarsan- 
ized serum.  Swift  and  Ellis  injected  salvarsanized  serum  intrathecally 
in  a  number  of  cases  of  tabes  dorsalis  and  in  other  manifestations  of 
neurosyphilis,  and  reported  most  encouraging  results  in  both  clinical 
and  immunological  manifestations.  This  work  has  since  been  confirmed 
by  a  large  number  of  authors.  The  treatment  is  of  special  value  in  the 
earlier  stages  of  neurosyphilis.  Unfavorable  results  have  been  ob- 
served, as  for  instance  the  spasmodic  retention  of  urine.  As  a  result 
of  long  standing  of  the  salvarsanized  serum  prior  to  its  use,  the  drug 
may  become  oxidized  with  a  marked  increase  in  toxicity. 

Method  of  Treatment. — Six-tenths  to  nine-tenths  gram  of  salvar- 
san or  neosalvarsan  is  injected  intravenously.  One  hour  later  40  c.c.  of 
the  patient's  blood  is  withdrawn,  allowed  to  coagulate  and  centrifuged. 
Twelve  cubic  centimetres  of  the  sterile  serum  is  diluted  with  18  c.c.  of 
sterile  physiological  salt  solution  to  make  it  a  40  per  cent,  dilution  and 
heated  for  half  an  hour  at  56°  C.  A  lumbar  puncture  is  then  per- 
formed, and  25  to  30  c.c.  of  fluid  is  withdrawn,  and  the  serum  very 
slowly  injected.  Swift  and  Ellis  recommend  the  gravitation  method  of 
injection  to  prevent  a  sudden  increase  in  intrathecal  pressure.  The 
patient  is  then  kept  in  bed  for  twenty-four  hours  and  the  foot  of  the 
bed  elevated  for  part  of  this  time.  The  reaction  is  usually  of  a  mild 


266  THE  PRINCIPLES  OF  IMMUNOLOGY 

type,  including  slight  fever,  pain  in  the  legs,  but  in  rare  instances  violent 
symptoms  have  been  observed.  Before  and  after  the  treatment  a  Was- 
permann  test,  the  globulin  test  and  a  cell  count  should  be  made.  After 
one  week  or  more  the  treatment  can  be  safely  repeated  until  definite 
improvement  occurs. 

TREATMENT  WITH  IMMUNE  HUMAN  SERUM 

Weisbecker  in  1897  appeared  to  be  the  first  to  have  used  blood 
serum  of  convalescents,  in  cases  of  scarlet  fever,  but  with  little  success. 
Huber  and  Blumenthal,  von  Leyden  and  others  renewed  the  interest 
in  convalescent  serum  therapy  but  failed  to  reach  any  definite  con- 
clusion probably  because  of  the  small  doses  employed.  Reiss  and 
Jungmann,  Koch,  Zingher  and  Weaver  more  recently  applied  the 
treatment  with  a  fair  degree  of  success.  Reiss  and  Jungmann  gave 
intravenous  injections  of  40  c.c.  to  100  c.c.  and  drew  the  blood  from 
scarlet-fever  convalescents  about  the  end  of  the  third  or  beginning  of  the 
fourth  week  of  the  disease,  testing  each  serum  for  the  possibility  of 
syphilis  and  for  sterility.  Zingher  injected  citrated  whole  blood  intra- 
muscularly in  doses  of  120  c.c.  to  240  c.c.  and  repeated  in  two  or  three 
days  if  necessary.  Weaver  drew  the  blood  from  convalescents  between 
the  twentieth  to  twenty-eighth  day,  only  such  convalescents  being  selected 
who  had  not  been  septic  and  who  gave  a  negative  Wassermann  reaction. 
The  sera  were  tested  for  sterility  and  used  pooled.  Intramuscular  in- 
jections were  given  in  doses  of  25  c.c.  to  90  c.c.,  60  c.c.  being  the  usual 
amount.  The  effects  of  the  serum  are  rapid  and  start  with  a  sudden  drop 
in  temperature  and  general  improvement  of  the  patient  within  twenty- 
four  hours  after  the  administration  of  the  serum.  The  best  results 
are  obtained  when  the  patients  are  treated  early  in  the  disease.  Kling 
and  Widfeldt  also  reported  favorable  results  in  their  series  of  cases 
during  an  epidemic  of  237  cases  at  Stockholm  in  1918.  This  method  has 
not  been  widely  adopted  and  there  is  still  much  question  as  to  whether 
improvement  is  due  to  the  treatment  or  to  the  natural  self -limitation 
of  the  disease. 

Monvoisin  has  recently  reported  encouraging  results  in  typhus  fever 
by  intravenous  injections  of  human  convalescent  serum.  One  or  two 
cubic  centimetres  of  serum  brought  a  marked  drop  in  temperature  and 
general  improvement  in  the  patient.  Monvoisin  noted  a  decrease  in 
mortality  from  30  down  to  10.34  per  cent,  by  the  use  of  convalescent 
sera.  The  serum  was  obtained  from  a  patient  on  the  eighth  day  after 
subsidence  of  fever.  Favorable  results  were  also  reported  by  Teissier 
in  cases  of  severe  and  hemorrhagic  smallpox.  In  leprosy  the  serum 
obtained  from  cantharides  blisters  on  lepers  has  been  reported  to 
be  of  value. 

Bleyer  recently  injected  immune  human  blood  into  a  series  of  forty- 
five  cases  of  whooping-cough  in  the  early  weeks  of  the  disease.  This 
series  was  divided  into  three  groups.  The  first  group  received  blood 
from  persons  who  were  convalescent  or  who  had  recovered  from 
whooping-cough  within  three  months.  In  the  second  series  the  blood 


EMPLOYMENT  OF  BLOOD  SERUM  267 

was  from  persons  who  had  the  disease  at  more  remote  periods,  and  the 
third  group  from  persons  who,  so  far  as  they  knew,  had  never  suffered 
with  whooping-cough.  The  stage  at  which  the  treatment  was  given 
was  about  the  same  in  the  three  groups  and  the  dosage  depended  upon 
the  body  weight  of  the  patient,  varying  between  40  c.c.  and  125  c.c., 
divided  into  two,  three  or  four  doses  and  injected  into  the  gluteus 
muscles.  In  the  first  group  there  were  no  deaths  and  no  complications, 
and  the  course  of  the  disease  was  in  no  definite  way  different  from 
the  usual  course.  The  second  group  showed  quite  as  satisfactory  im- 
provement as  in  the  first  group.  In  the  third  group  there  were  two 
pneumonia  cases  with  one  death  and  one  case  which  apparently  was 
favorably  influenced  by  normal  serum  treatment.  The  groups  are  so  small 
and  the  difference  so  slight  as  to  give  no  reason  for  regarding  this 
mode  of  treatment  as  particularly  effective.  Vaccine  treatment  of  this 
disease  gives  much  greater  promise  of  success. 

During  the  recent  great  epidemics  of  so-called  influenza,  conva- 
lescent serum  was  used  in  a  considerable  number  of  cases  which 
developed  pneumonia.  In  many  instances  there  was  marked  improve- 
ment, but  there  is  no  clear  indication  that  the  results  were  specific  or 
that  they  depended  absolutely  upon  the  serum  treatment. 

SERUM  THERAPY  IN  INFECTIONS  OF  UNDETERMINED  ETIOLOGY 

Introduction. — The  preparation  of  the  immune  sera  discussed  above 
depends  not  only  upon  knowledge  of  the  etiological  agent  of  the  disease 
concerned  but  also  necessitates  the  isolation  of  the  organism  in  pure 
culture.  Several  infectious  agents  are  known  to  exist  in  blood  and 
tissues,  since  the  diseases  may  be  transmitted  by  means  of  inoculation 
of  blood,  organs  or  organ  extracts.  Many  of  these  agents  are  so  small 
as  to  pass  through  porcelain  filters  and  are  spoken  of  as  the  filterable 
viruses.  Some  of  these  viruses  have  been  observed  to  contain  minute 
globoid  bodies  which  have  been  obtained  in  pure  culture,  but  under 
such  conditions  that  they  have  not  served  well  as  antigens  for  the 
production  of  immune  sera.  If  immunization  be  attempted  by  injec- 
tions of  the  blood  or  tissues  containing  the  infective  agents,  the  result- 
ing immune  serum  contains  not  only  antibodies  for  the  infective  agent 
but  also  for  the  tissues.  If  these  tissues  happen  to  be  from  the  same 
species  into  which  the  serum  is  to  be  injected  the  hemagglutinins, 
hemolysins  and  cytolysins  in  the  immune  serum  may  seriously  damage 
or  even  kill  the  individual  so  treated.  Active  immunization  by  the  use 
of  infected  tissues  appears  to  progress  favorably  in  spite  of  the  presence 
of  the  tissues,  as  seen  in  the  active  immunization  of  man  and  other 
animals  by  the  use  of  the  virus  of  rabies*  contained  in  the  dried  spinal 
cords  of  rabbits.  It  is  in  the  production  of  sera  for  passive  immuniza- 
tion that  the  danger  from  simultaneously  formed  tissue  antibodies  ap- 
pears. Rous,  Robertson  and  Oliver  have  studied  this  problem  with  a 
view  to  removing  from  the  immune  serum  these  harmful  elements. 
After  the  immune  serum  is  prepared  the  tissue  antibodies  are  removed 
by  selective  absorption  with  red  blood-corpuscles,  since  these  cells  re- 


268  THE  PRINCIPLES  OF  IMMUNOLOGY 

move  the  most  important  source  of  danger,  the  hemagglutinins  and  the 
hemolysins;  undoubtedly  many  of  the  other  tissue  antibodies,  as  the 
cytolysins,  are  reduced  in  amount.  For  example,  they  immunized  a 
goat  with  megatheriolysin  and  finely-ground  liver,  spleen  and  kidney, 
as  well  as  defibrinated  blood,  of  guinea-pigs.  The  immune  serum  was 
then  repeatedly  mixed  with  guinea-pig  blood-cells  until  all  the  hemag- 
glutinin  and  hemolysin  had  been  removed.  The  process  did  not  reduce 
the  titer  of  the  special  antilysin  against  megatheriolysin  either  in  test 
tube  or  animal  experiments.  Guinea-pigs  were  protected  against 
megatheriolysin  by  the  use  of  this  serum  and  the  treatment  of  the 
serum  by  selective  absorption  removed  practically  all  the  elements  dan- 
gerous for  the  guinea-pig.  Similar  experiments  were  performed  using 
as  antigen  the  blood  of  rabbits  suffering  from  pneumococcus 
septicemia.  It  was  found  that  absorption,  by  means  of  blood,  of 
anti-poliomyelitis  serum  produced  no  change  in  its  protective  value. 
Experiments  were  also  performed  with  the  Rous  chicken  sarcoma,  a 
tumor  caused  by  a  filterable  virus.  The  immune  serum  was  prepared 
by  injecting  into  geese  a  mixture  of  tumor  tissue  and  the  blood  of 
moribund  fowl  since  under  these  circumstances  the  blood  contains  the 
causative  agent.  The  immune  serum  was  treated  with  fowl  blood- 
corpuscles  to  remove  the  tissue  antibodies.  The  serum  so  treated,  when 
employed  in  proper  ratio  to  the  amount  of  tumor  inoculated,  served 
to  protect  fowl  against  the  subsequent  growth  and  development  of  the 
tumor,  whereas  growth  proceeded  regularly  in  the  unprotected  con- 
trols. Rous  makes  no  claim  as  to  high  protective  value  but  that  some 
such  power  is  developed  is  undoubted. 

The  work  quoted  above  is  of  the  utmost  importance  in  establishing 
the  important  principles  that  must  be  observed  in  the  preparation  of 
immune  sera  against  infective  agents  either  known  or  unknown  when 
used  as  antigens  in  animal  tissues.  The  studies  are  recent  and  have  not 
as  yet  been  widely  applied.  The  immune  sera  against  infections  of 
undetermined  cause  to  be  described  in  this  section  were  studied  before 
the  work  of  Rous  and  his  associates  appeared,  and  it  is  probable  that 
the  methods  of  preparation  may  be  considerably  modified  in  the  course 
of  time.  The  inclusion  of  acute  anterior  poliomyelitis  in  this  group 
is  justified  only  on  the  ground  of  dissension  as  to  whether  the  disease 
is  due  to  the  globoid  bodies  described  by  Flexner  and  his  collaborators 
or  to  the  pleomorphic  streptococcus  studied  by  Rosenow,  Nuzum 
and  others. 

Anti-poliomyelitis  Serum. — That  one  attack  of  poliomyelitis  pro- 
tects against  subsequent  infection  has  been  known  for  many  years. 
Levaditi  and  Landsteiner  and  also  Flexner  and  Lewis  in  1910 
demonstrated  that  the  serum  of  convalescents  and  of  monkeys  recov- 
ered from  the  disease  protects  against  infection.  Treatment  of  human 
cases  of  the  disease  was  applied  by  Netter  in  1916.  This  author 
injected  intrathecally  the  serum  of  recovered  patients  in  doses  of  5  to 
13  c.c.  for  a  period  of  eight  days  with  most  encouraging  results.  He 
believed  that  the  best  serum  is  found  in  individuals  whose  acute  attack 


EMPLOYMENT  OF  BLOOD  SERUM  269 

dates  back  from  three  months  to  four  years.  Flexner  carried  out 
experiments  with  monkeys  and  proved  that  the  serum  of  recovered 
cases  was  efficacious  in  the  cure  of  these  animals.  In  1916-1917  this 
author  used  the  serum  extensively  during  the  epidemic  in  New  York 
and  recommends  the  combination  of  intraspinal  and  intravenous  in- 
jections. Children  were  given  combined  doses  of  5  to  10  c.c.  intra- 
spinally  and  30  to  40  c.c.  intravenously.  The  possibility  of  conveying 
the  disease  is  not  considered  a  danger,  because  the  virus  has  never  been 
detected  in  the  blood.  The  only  difficulty  encountered  in  this  method 
of  treatment  is  that  of  securing  sufficient  quantities  of  serum.  Pooling 
of  sera  is  of  the  greatest  advantage,  since  the  antibody  content  may 
vary  widely  in  the  sera  of  different  persons. 

During  the  epidemic  of  1917  Mathers,  Rosenow,  Towne  and 
Wheeler,  Nuzum  and  Herzog,  and  later  Nuzum  reported  the  discovery 
of  a  pleomorphic  streptococcus  which  they  had  constantly  observed  in 
the  brain  and  spinal  cord,  and  also  in  the  cerebrospinal  fluid  in  human 
cases  of  poliomyelitis.  Flexner  and  Noguchi,  Smillie  and  many  others 
deny  the  etiological  importance  of  this  streptococcus.  Rosenow,  Nuzum 
and  Willy  claim  to  have  produced  sera  with  definite  protective  and  cura- 
tive effects.  In  the  hands  of  Nuzum  and  Willy  serum  treatment  re- 
duced the  mortality  in  a  series  of  159  cases  from  38  per  cent,  to 
11.9  percent. 

Amoss  reported  that  only  imperfect  success  in  developing  antibodies 
in  rabbits  and  monkeys  has  attended  the  repeated  injection  of  cultures 
of  the  globoid  bodies  of  Flexner  and  Noguchi  and  also  failed  to  find 
evidence  that  Rosenow's  serum  is  either  therapeutically  effective  in 
monkeys  or  possesses  antibodies  of  the  same  nature  as  those  present  in 
the  blood  of  monkeys  which  have  recovered  from  experimental  polio- 
myelitis. Since  the  antibodies  in  convalescent  poliomyelitis  serum  in  man 
and  monkey  are  identical,  this  author  states  that  any  antibodies  present 
in  Rosenow's  horse  serum  do  not  conform  to  those  occurring  in  human 
convalescent  serum.  Again  Amoss  and  Eberson  in  a  later  paper  con- 
cluded that  the  anti-streptococcus  serum  of  Nuzum  and  Willy  fails  to 
show  in  the  monkey  neutralizing  or  therapeutic  power  against  small 
doses  of  the  virus  of  poliomyelitis.  Under  the  same  conditions  the 
serum  of  monkeys  which  had  recovered  from  experimental  poliomye- 
litis proved  neutralizing  and  protective.  These  facts  leave  some  doubt 
as  to  the  actual  value  of  anti-poliomyelitis  horse  serum,  and  until 
more  conclusive  evidence  has  been  brought  forward  by  the  supporters 
of  the  streptococcus  as  an  etiological  factor  we  believe  that  the  only 
effective  serum  existing  is  that  of  convalescent  or  recovered  cases. 
Neustadter  and  Banzhaf  immunized  horses  against  a  filtrate  obtained 
from  the  digested  brain  and  cord  of  a  human  case  of  the  disease.  The 
immune  serum  gave  encouraging  results  in  a  few  experiments  with 
monkeys,  but  as  yet  data  are  too  limited  to  justify  a  conclusion  as  to 
the  usefulness  of  this  serum. 

Rinderpest. — Kolle  and  Turner  injected  gradually  increasing  doses 
of  virulent  rinderpest  blood  and  also'  bile  of  infected  animals  into  oxen 


270  THE  PRINCIPLES  OF  IMMUNOLOGY 

and  obtained  potent  sera  against  the  rinderpest  virus.  Of  3318  animals 
treated  with  this  serum  455  or  13.9  per  cent,  died,  while  the  mortality 
among  non-treated  animals  averages  between  85  per  cent,  and  95  per 
cent.  The  serum  can  be  used  prophylactically  in  doses  of  100  to  200 
c.c.  If  the  virus  is  simultaneously  injected  in  small  doses  as  advised  ' 
by  these  authors,  the  results  appear  to  be  extremely  satisfactory. 
The  serum  for  curative  purposes  should  be  employed  within  thirty  days 
after  the  onset  of  fever. 

Anti-hog-cholera  Serum. — Immunization  against  hog  cholera  has 
an  important  historical  as  well  as  a  practical  bearing  since  it  was  in  this 
disease  that  the  first  attempt  to  immunize  with  bacterial  products  was 
made.  Salmon  and  Theobald  Smith  published  in  1884  their  account 
of  the  production  of  immune  sera  in  the  pigeon  by  the  inoculation  of 
killed  broth  culture  of  the  bacillus  of  hog  cholera.  Subsequent  studies 
have  made  it  appear  that  the  disease  is  not  due  to  the  bacillus  of  hog 
cholera  and  much  evidence  is  at  hand  to  support  the  view  that  the 
etiological  agent  is  a  filterable  virus.  At  the  present  time  immunity  is 
produced  in  healthy  hogs  by  the  injection  of  blood  obtained  from 
infected  hogs,  thus  implanting  the  virus.  It  is  necessary  to  protect 
the  animals  employed  by  passive  immunization  with  a  previously- 
prepared  antiserum.  The  animals  selected  are  injected  subcutaneously 
with  40  c.c.  of  anti-hog-cholera  serum  per  hundred  pounds  of  weight. 
Two  to  three  days  later  the  animals  receive  intravenously  3  or  4  c.c. 
of  defibrinated  blood  obtained  from  an  animal  suffering  from  the 
disease,  or  the  animals  may  be  exposed  in  infected  pens.  If  the  animals 
survive,  after  a  period  of  one  month  they  are  given  5  c.c.  of  the  living 
virus.  This  is  repeated  after  two  or  three  weeks.  The  immunized 
animals  are  bled  from  the  tail.  Five  cubic  centimeters  of  blood  per 
pound  of  weight  are  usually  withdrawn.  The  protective  power  of  the 
serum  thus  obtained  is  then  determined  in  a  series  of  hogs.  For 
prophylactic  purposes  the"  animals  receive  40  c.c.  subcutaneously  per 
hundred  pounds  of  weight  or  simultaneous  injections  of  virus  and 
serum,  but  this  combination  is  not  without  danger.  For  therapeutic 
purposes  several  injections  are  necessary  and  the  serum  should  be 
administered  as  early  as  possible. 

THERAPEUTIC  USE  OF  NORMAL  SERUM 

Normal  serum  therapy  in  man  has  included  the  use  of  both  human 
and  animal  sera.  In  the  treatment  of  natural  or  experimental  disease 
in  man  or  animals  the  normal  serum  employed  may  be  homologous  or 
heterologous.  The  basis  of  such  method  of  treatment  has  often  been 
entirely  empirical,  but  as  serum  therapy  has  been  more  carefully 
studied  the  employment  of  normal  serum  may  be  placed  in  two  cate- 
gories, namely  that  of  the  non-specific  protein  treatment  of  disease 
or  that  of  providing  the  blood  with  certain  elements  necessary  for  the 
process  of  clotting.  It  is  to  be  conceded  that  a  normal  serum  may  be 
employed  because  of  some  natural  antibodies  which  it  may  contain, 
but  such  a  form  of  passive  immunization  is  much  improved  if  the 


EMPLOYMENT  OF  BLOOD  SERUM  271 

natural  antibodies  are  increased  by  specific  immunization.  The  use  of 
normal  serum  in  non-specific  therapy  probably  increases  those  non- 
specific factors  of  defense  such  as  fever,  serum  enzymes,  etc.,  that 
have  already  been  discussed.  In  hemophilia,  purpura  hemorrhagica, 
melena  neonatorum  and  similar  hemorrhagic  diseases  there  is  a  disturb- 
ance of  proper  balance  of  those  constituents  of  the  blood  and  tissues 
which  provide  for  coagulation  of  the  blood.  Hypotheses  differ  as  to 
the  exact  mechanism  of  the  process  of  coagulation,  but  fundamentally 
it  seems  necessary  to  have  an  equilibrium  of  prothrombin  and  anti- 
thrombin.  This  balance  may  be  upset  by  an  excess  of  antithrombin, 
by  a  deficiency  in  prothrombin,  fibrinogen,  calcium  salts  or  other  ele- 
ments. The  interaction  of  prothrombin,  thrombokinase  (or  thrombo- 
plastin)  and  calcium  salts  results  in  the  formation  of  thrombin. 
Thrombin  and  fibrinogen  interact  to  form  fibrin,  the  essential  element 
of  a  clot.  Blood  serum  is  rich  in  prothrombin  and  if  a  hemorrhagic 
disease  be  due  to  prothrombin  deficiency,  serum  treatment  is  likely  to 
be  beneficial.  If,  on  the  other  hand,  the  disease  be  due  to  an  excess 
of  antithrombin  the  introduction  of  prothrombin  has  little  value. 
Similarly  hemorrhagic  disease  with  low  fibrinogen  content  is  not  bene- 
fited by  serum  treatment.  Whipple  has  found  decrease  of  fibrinogen 
in  advanced  cirrhosis  of  the  liver  with  hemorrhage,  excess  of  antithrom- 
bin in  aplastic  anemia  and  leucemia  and  deficiency  of  prothrombin 
in  melena  neonatorum.  Duke  holds  that  the  lack  of  prothrombin 
is  due  to  a  deficiency  in  the  number  of  platelets,  whereas  Minot 
and  Lee  believe  that  in  hemophilia,  at  least,  the  slow  clotting  is  due 
to  a  hereditary  defect  in  the  platelets  which  renders  them  less  avail- 
able for  the  process  of  coagulation.  Various  studies  have  given  different 
results  as  to  the  changes  found  in  the  elements  concerned  in  clotting. 
Whipple  points  out  that  if  the  phenomenon  is  studied  in  the  individual 
case  rational  therapy  may  be  applied.  In  melena  neonatorum  the  ad- 
ministration of  blood  serum  often  gives  brilliant  results.  In  other 
hemorrhagic  diseases  the  results  are  somewhat  more  variable.  If 
hemorrhage  has  been  severe  and  anemia  is  marked,  the  double  purpose 
of  favoring  clotting  and  replacing  lost  blood  may  be  served  by  trans- 
fusion from  a  suitable  and  properly-tested  donor.  The  more  direct 
the  transfusion  the  less  likelihood  is  there  of  alteration  of  the  blood 
due  to  beginning  clotting  and  the  greater  is  the  probability  of  con- 
tributing substances  to  replace  or  augment  those  which  may  be  deficient 
in  the  patient's  blood. 


APPENDIX  B 

PROPHYLACTIC  VACCINATION 

INTRODUCTION. 

TYPES  OF  VACCINES. 

LIVING  VACCINES. 
SENSITIZED  VACCINES. 
KILLED  BACTERIAL  VACCINES. 
PREPARATION  OF  BACTERIAL  VACCINES. 
METHODS  OF  COUNTING. 

HEMOCYTOMETER  METHOD. 
WRIGHT'S  METHOD. 
OTHER  METHODS. 
DOSAGE  OF  ORGANISMS. 
LIPOVACCINES. 
CONTRAINDICATIONS. 
VACCINATION  WITH  LIVING  VIRUS. 
SMALL-POX  VACCINATION. 
PREPARATION. 
METHODS  OF  INOCULATION. 
LINEAR  INCISION. 
DRILL  METHOD. 
MULTIPLE  PUNCTURE. 
INTRACUTANEOUS  METHOD. 
VACCINIA. 

IMMUNITY  AS  THE  RESULT  OF  VACCINATION. 
UNFAVORABLE  RESULTS  OF  VACCINATION. 
RABIES  VACCINATION. 

ACTIVE  IMMUNIZATION  (VACCINATION). 
PREPARATION  OF  MATERIAL. 
VACCINATION  IN  MAN. 
EFFECTS  OF  VACCINATION. 
PROTECTIVE  RESULTS. 
VACCINATION  WITH  KILLED  ORGANISMS. 
TYPHOID  AND  PARATYPHOID  FEVERS. 
PREPARATION  OF  VACCINES. 
METHOD  OF  ADMINISTRATION. 
PROPHYLACTIC  VALUE. 
DURATION  OF  PROTECTION. 
COMPLICATIONS. 
CONTRAINDICATIONS. 

CHOLERA. 
PNEUMONIA. 
PLAGUE. 
TYPHUS  FEVER. 
PERTUSSIS. 
DYSENTERY. 
INFLUENZA. 
OTHER  DISEASES. 

VACCINATION 

Introduction. — In  contrast  to  the  methods  of  passive  immunization, 
i.e.,  the  parenteral  introduction  of  immune  sera,  vaccine  treatment  aims 
to  increase  the  resistance  to  disease  by  the  injection  of  the  causal 
organisms  or  their  products.  The  duration  of  this  increased  resistance 
varies  in  time  according  to  species  and  types  of  organisms  injected  and 
the  individual  characterisics  of  the  subject.  For  instance,  vaccination 
272 


PROPHYLACTIC  VACCINATION  273 

against  smallpox  immunization  may  last  for  a  considerable  number  of 
years,  while  with  other  organisms,  such  as  the  staphylococcus  or  pneu- 
mococcus  the  immunity  is  of  relatively  short  duration. 

The  aims  of  vaccination  are  either  to  cause  prophylactic  resistance 
against  disease  or  to  increase  an  already  established  resistance.  Proph- 
ylactic vaccination  against  typhoid  is  an  example  of  the  former,  while 
the  vaccine  treatment  of  furunculosis  or  gonorrhea  are  examples 
of  the  latter. 

The  term  vaccine  is  derived  from  vaccinia  or  cowpox,  and  the  method 
of  protective  immunization  against  smallpox  with  vaccinia  virus  was 
called  by  Jenner  "  vaccination."  This  great  empirical  work  was  placed 
on  a  sound  scientific  basis  by  Pasteur  after  he  had  discovered  the 
method  of  protective  inoculation  against  chicken  cholera,  and  Pasteur 
used  the  term  vaccination  for  such  inoculations.  To-day  the  simple 
term  vaccine  is  loosely  applied  and  should  be  restricted  to  cowpox 
vaccine.  Suspensions  of  bacteria  such  as  typhoid  bacilli  or  pyogenic 
cocci  should  be  designated  bacterial  vaccines.  Wright  defines  a  bac- 
terial vaccine  as  follows  :  "  Bacterial  vaccines  are  sterilized  and  enumer- 
ated suspensions  of  bacteria  which  furnish,  when  they  dissolve  in  the 
body,  substances  which  stimulate  the  healthy  tissues  to  the  production 
of  specific  bacteriotropic  substances  (or  antibodies)  which  fasten 
upon  and  directly  or  indirectly  contribute  to  the  destruction  of  the 
corresponding  bacteria." 

Perhaps  the  first  serious  attempt  to  apply  practically  a  bacterial 
vaccine  in  the  treatment  of  human  disease  was  that  of  Koch,  who  in 
1890  employed  tuberculin  in  the  treatment  of  tuberculosis.  In  1893 
Frankel  treated  thirty-seven  cases  of  typhoid  fever  with  subcutaneous 
injections  of  killed  typhoid  bacilli.  He  reported  that  the  course  of 
the  disease  was  favorably  modified  and  in  a  few  instances  terminated 
by  rapid  lysis.  Rumpf  treated  a  series  of  cases  of  typhoid  fever  with 
bacillus  pyocyaneus  and  obtained  equally  favorable  results,  thus  throw- 
ing doubt  upon  the  specific  character  of  the  treatment  and  leading  into 
the  newer  field  of  non-specific  therapy.  Wright  and  Douglas  soon 
after  the  discovery  of  opsonins  demonstrated  their  method  of  treatment 
by  bacterial  vaccines  under  the  guidance  of  the  opsonic  index.  Wright 
stated  that  a  patient  who  had  become  infected  by  an  organism  such  as 
the  staphylococcus  aureus  or  the  tubercle  bacillus  would  be  found  to 
have  a  lowered  resistance  against  these  organisms;  that  this  degree 
of  want  of  resistance  could  be  accurately  determined,  and  that  the 
resistance  could  be  stimulated  and  controlled  by  measured  doses  of 
a  vaccine  of  the  causative  organism.  Wright's  method  of  treatment 
was  based  on  the  principle  of  strict  specificity.  It  was  soon  pointed  out 
that  opsonins  are  only  one  link  in  the  defensive  chain  of  the  host,  and 
the  use  of  the  method  has  been  somewhat  restricted.  The  measure  of 
opsonins  in  a  given  instance  was  subsequently  found  not  to  be  a  measure 
of  the  existing  degree  of  total  immunity.  In  the  majority  of  diseases, 
therapeutic  vaccination  has  not  withstood  the  test  of  time.  Wright 
himself,  after  experiences  in  the  World  War,  stated  that  it  has  been 
18 


274  THE  PRINCIPLES  OF  IMMUNOLOGY 

accepted  that  the  inoculation  of  microbes  into  the  already  infected 
system  is  as  illogical  as  to  instil  further  poison  into  an  already  poisoned 
body.  However,  a  wide  field  for  prophylactic  vaccination  is  still  open. 
Soon  after  Wright's  work  bacterial  vaccines  were  applied  in  every 
conceivable  way  and  unfortunately  much  harm  has  been  done  to  the 
rational  use  of  vaccines  by  reckless  commercialism. 

Wright  and  his  collaborators  have  studied  carefully  the  opsonic 
index  of  patients  the  victims  of  infectious  disease  as  well  as  that  of 
normal  individuals.  They  found  that  phagocytosis  is  often  depressed 
in  those  who  are  unsuccessfully  combating  certain  disease  and  that  the 
phagocytic  power  can  be  increased  by  specific  bacterial  vaccination. 
They  pointed  out  further  that  following  the  first  dose  of  vaccine  the 
opsonic  index  is  considerably  depressed  and  spoke  of  this  phenomenon 
as  the  negative  phase.  This  phase  may  last  for  several  days  and 
numerous  writers  have  thought  that  such  a  depression  of  phagocytic 
resistance  might  indicate  such  a  decrease  of  general  immunity  as  to 
render  vaccination  during  an  epidemic  highly  undesirable.  The  nega- 
tive phase  has  been  carefully  investigated  and  many  now  believe  that 
it  does  not  exist.  The  factor  of  error  in  the  determination  of  the 
opsonic  index  is  considerable,  owing  to  the  variability  of  conditions 
operating  in  vitro.  Therefore,  it  is  possible  that  the  decrease  of  index 
pointed  to  by  Wright  may  fall  within  the  limit  of  experimental  error. 
The  recent  observations  of  Balteano  and  Lupu  indicate  that  no  such 
negative  phase  is  demonstrable  in  cholera,  and  the  careful  investiga- 
tions of  Cantacuzene  indicate  that  the  negative  phase  does  not  occur 
in  other  diseases. 

Types  of  Vaccines. — Living  Vaccines. — From  animal  experiments 
it  is  generally  admitted  that  the  greatest  and  most  lasting  immunity  is 
produced  by  the  injection  of  living  bacteria.  The  killing  of  bacteria 
apparently  destroys  certain  thermolabile  substances  which  possess  anti- 
genie  properties.  In  human  practice  the  use  of  living  bacteria  is  not 
without  danger.  One  may  at  first  inoculate  with  a  single  living  organ- 
ism and  cautiously  increase  the  number,  but  the  virulence  of  the  organ- 
ism is  not  easily  controlled  and  may  be  so  great  as  to  make  such 
inoculations  dangerous.  In  addition  there  is  a  risk  of  establishing  a 
"  carrier  state  "  since  the  gradual  increase  of  the  number  of  organisms 
may  establish  a  mutual  immunity  on  the  part  of  both  the  parasite  and 
the  host.  If  the  virus  of  the  disease  can  be  so  attenuated  that  danger 
of  producing  an  outspoken  attack  of  the  disease  is  eliminated,  vac- 
cination can  be  performed  with  great  success.  The  outstanding  ex- 
amples of  this  method  in  human  medicine  are  vaccination  against 
smallpox  and  against  rabies.  In  smallpox  the  virus  is  attenuated 
by  animal  passage  through  the  calf  and  in  rabies  the  virus  is  attenuated 
by  desiccation. 

Sensitised  Vaccines. — These  are  bacterial  vaccines  composed  of 
"bacteria  which  have  been  exposed  to  their  specific  immune  serum.  As 
early  as  1891  Babes  mixed  the  blood  of  a  highly  refractory  dog  with 
an  emulsion  of  street  virus  in  order  to  produce  in  other  animals  a  more 


PROPHYLACTIC  VACCINATION  275 

rapid  development  of  immunity  against  rabies.  In  the  only  experiment 
reported  at  this  time  it  was  shown  that  some  protection  was  afforded 
by  the  mixture,  although  the  inoculated  animal  finally  succumbed  to 
rabies.  Lorenz  in  1892  made  similar  observations  in  swine  erysipelas. 
Since  this  time  numerous  workers  have  used  the  method.  The  most 
important  advance  was  made  when  Besredka  suggested  the  removal 
of  the  excess  of  serum  by  centrifugally  washing  the  sensitized  bacteria. 
Subsequent  work  has  been  carried  on  with  killed  bacteria  treated  with 
their  immune  sera,  washed  and  suspended  in  a  suitable  menstruum. 
The  ordinary  non-sensitized  bacterial  vaccines  injected  into  an  animal 
during  the  incubation  period  of  a  disease  are  likely  to  hasten  the  death 
of  the  animal,  or  if  the  infection  is  already  acquired,  the  injection  of 
the  vaccine  appears  to  lower  the  natural  resistance.  Besredka  and 
Metchnikoff  believe  that  sensitized  bacterial  vaccines  produce  no  nega- 
tive phase,  but  only  slight  local  and  general  reactions  and  facilitate  the 
production  of  antibodies.  Kakechi  has  shown  that  the  toxicity  of 
sensitized  bacterial  vaccines  is  less  than  that  of  the  non-sensitized. 
Sensitized  bacterial  vaccines  have  been  employed  in  numerous  infec- 
tious diseases  such  as  typhoid  fever,  asiatic  cholera  and  bubonic  plague 
with  varying  degrees  of  success. 

Killed  Bacterial  Vaccines. — These  are  suspensions  of  bacteria  usu- 
ally in  salt  solution  but  sometimes  in  other  mentsrua  such  as  neutral 
oil.  The  organisms  are  usually  killed  after  the  suspension  has  been 
made,  but  in  making  oil  suspensions  the  organisms  are  killed  before 
the  final  suspension.  Heat  is  usually  employed  for  killing  the  bacteria 
and  the  action  is  further  supplemented  by  the  addition  of  a  bactericidal 
preservative  to  the  suspension.  Under  certain  circumstances  chemicals 
such  as  formaldehyde  or  phenol  may  be  employed  both  for  killing  and 
preserving  the  vaccine.  Autogenous  vaccines  are  bacterial  vaccines 
prepared  from  bacteria  which  have  been  freshly  isolated  from  the 
individual  patient.  At  times  it  is  very  difficult  to  isolate  the  organism  as 
for  instance  in  gonorrhea.  In  these  cases  stock  vaccines  are  usually 
employed.  Stock  vaccines  are  made  from  strains  of  bacteria  isolated 
at  some  previous  time  aad  kept  in  the  laboratory  stock.  Stock  vaccines 
are  used  extensively  in  prophylactic  vaccinations.  Mixed  vaccines  are 
composed  of  various  kinds  of  bacteria.  Their  value  is  questionable 
and  their  use  unscientific,  except  on  the  basis  of  non-specific  therapy. 
Many  efforts  have  been  made  to  produce  the  bacterial  antigen  in  a  pure 
form  so  as  to  obtain  a  minimum  of  local  and  general  reaction,  and  to 
immunize  in  the  shortest  space  of  time  possible.  Such  vaccines  have 
been  made  from  nucleoproteins,  autolyzed  bacteria,  digested  bacteria 
and  detoxicated  organisms.  It  appears  that  some  of  these  methods  are 
promising,  especially  for  the  production  of  antigens  from  spore- 
bearing  bacteria. 

Preparation  of  a  Bacterial  Vaccine. — Under  strict  asepsis  an  emulsion  of 
the  organism  in  question  is  prepared  by  adding  5  to  10  c.c.  of  physiological  salt 
solution  to  a  twenty-four-hour  agar  slant  culture.  This  is  allowed  to  stand  ten 
minutes  and  then  rotated  actively  in  order  to  make  a  suspension  of  the  organisms. 
The  suspension  is  now  filtered  through  sterilized  filter  paper  in  a  funnel  into  a 


276  THE  PRINCIPLES  OF  IMMUNOLOGY 

sterile  test-tube.  In  case  of  scanty  growth  the  emulsion  is  directly  transferred 
to  another  surface  culture,  the  growth  in  this  tube  suspended  and  the  process 
repeated  with  additional  growths  until  a  satisfactory  'emulsion  is  obtained. 
Instead  of  filtering  the  emulsion  one  may  shake  the  emulsion  in  a  test  tube 
containing  glass  beads  to  break  up  the  clumps.  It  is  of  great  importance  to  have 
a  homogeneous  suspension.  Because  of  the  presence  of  pepton  or  proteins 
from  the  culture  media  some  authors  advise  washing  of  the  organisms  until  the 
supernatant  fluid  gives  a  negative  biuret  reaction.  The  next  step  in  the  prepara- 
tion is  the  counting  of  the  -emulsion.  This  can  be  done  by  the  hemocytometer 
method,  by  Wright's  method  and  other  methods. 

Hemocytometer  Method. — (From  Zinsser,  Hopkins  and  Ottenberg,  "  A 
Laboratory  Course  in  Serum  Study.") — A  staining  solution  is  prepared  by  adding 
to  20  c.c.  of  i  per  cent,  phenol  i  c.c.  of  a  saturated  alcoholic  solkition  of  thionin. 
A  small  amount  of  the  carefully  shaken  bacterial  suspension  is  removed  to  a 
watch  glass.  A  dilution  of  i-ioo  is  prepared  in  a  red  cell  pipette  with  the  staining 
solution  as  diluent  to  the  101  mark.  After  carefully  shaking  and  after  blowing 
out  the  portion  of  the  fluid  in  the  capillary  end  of  the  pipette  a  small  drop  is 
placed  in  a  counting  chamber  and  covered  with  a  flat  coverslip.  After  allowing 
fifteen  minutes  for  the  bacteria  to  settle  a  count  is  made,  with  4.  m,m.  objective, 
of  a  number  of  squares  until  200  or  more  bacteria  have  been  counted.  It  is  best 
to  take  this  count  from  different  portions  of  the  ruled  surface  and  from  two 
separate  drops  of  the  mixture.  The  small  squares  have  an  area  of  1/400  of  a 
square  mm.,  the  depth  of  the  chamber  is  o.i  mm.,  the  dilution  is  l-ioo.  The 
number  of  bacteria  may  be  estimated  by  the  following  formula : 

Number  of  bacteria  counted  X  400  X  10  X  100  X  1000 

— 3 . =  number  of  bacteria  in  i.o  c.c. 

Number  of  squares  counted 

Wright's  Method. — A  drawn-out  capillary  pipette  is  prepared  and  marked 
with  a  grease  pencil  about  2.  cm.  from  the  tip.  A  small  puncture  is  made  in  the 
tip  of  the  finger  and  a  fresh  drop  of  blood  obtained.  Three  units  of  salt  solution 
are  then  drawn  up  in  the  pipette,  admitting  a  bubble  of  air  between  each  unit  of 
salt  solution.  The  unit  is  the  amount  that  is  drawn  up  to  the  mark  on  the 
pipette.  Blood  from  the  finger-tip  is  then  drawn  up  to  the  mark,  a  bubble  of  air 
admitted  and  the  bacterial  suspension  drawn  up  to  the  mark.  The  mixture  is  then 
blown  out  on  a  clean  slide  and  drawn  in  and  out  of  the  pipette  several  times  to 
ensure  even  mixing  of  the  blood  and  bacteria.  A  drop  of  this  mixture  is  placed 
on  a  second  slide  and  carefully  spread  across  the  slide  in  the  manner  of  making 
blood  smears.  It  is  important  that  the  film  be  thin  and  even,  so  that  the  ^  red 
cells  are  not  piled  in  masses  in  any  portion  of  the  film.  This  film  is  stained 
with  Wright's  stain,  or  by  any  other  simple  method,  and  a  differential  count  of  the 
number  of  bacteria  and  red  cells  in  a  number  of  fields  in  different  parts  of  the 
slide  is  made.  For  this  a  rule  scale  to  be  inserted  in  the  eyepiece  of  the  micro- 
scope is  very  helpful.  Fields  are  counted  until  200  red  cells  have  been  counted. 
The  number  of  bacteria  in  the  suspension  may  then  be  estimated  from  the  number 
of  bacteria  counted,  using  the  following  formula  (assuming  that  the  blood  of  the 
worker  contains  5.000,000  red  cells  per  cmm.)  : 

Number  of  bacteria  X  5,000,000  X  i.ooo      XT  e  , 

=r= — r P ; — TS — 7 v — =  Number  of  bacteria  per  c.c. 

Number  of  red  cells  (200) 

Other  Methods. — Among  the  other  methods  of  standardization  of  the  sus- 
pension are  the  comparison  of  the  emulsion  with  a  known  standard  emulsion,  the 
estimate  of  the  average  number  of  organisms  per  slope  grown  in,  say,  eighteen 
hours,  or  an  estimate  of  the  number  of  germs  per  loopful  (Kolle's  method). 
Hopkins  centrifugalized  his  suspension  at  high  speed  in  a  special  tube  with 
graduated  tip  until  the  supernatant  fluid  was  clear.  The  number  of  organisms 
for  a  number  of  species  in  such  a  closely-packed  sediment  has  been  determined 
and  is  as  follows : 

Staphylococcus  aureus   o.oi  c.c.  equals  10     billion 

Streptococcus  hemolyticus   o.oi  c.c.  equals  8     billion 

Gonococcus o.oi  c.c.  equals  8     billion 

Pneumococcus  (capsulated)    o.oi  c.c.  equals  2.5  billion 

B.  typhosus o.oi  c.c.  equals  8     billion 

B.  coli o.oi  c.c.  equals  4      billion 


PROPHYLACTIC  VACCINATION  277 

Gates  recently  standardized  his  bacterial  suspension  by  measuring  the  opacity 
of  the  suspension  by  the  length  of  the  column  of  the  suspension  required  to  cause 
the  disappearance  of  a  wire  loop.  By  a  simple  formula  the  measured  opacity  is 
translated  into  terms  of  the  concentration  of  bacteria  per  cubic  centimeter  and  so 
made  comparable  with  that  of  other  suspensions  of  the  same  organism. 

The  stock  suspension  after  estimation  of  the  number  of  organisms  contained 
is  ready  for  dilution.  Shera  employs  the  following  method  for  dilution.  Suppose 
the  suspension  is  found  to  contain  6400  million  organisms  per  cubic  centimeter, 
and  that  a  vaccine  of  1000  millions  per  cubic  centimeters  is  required.  Five  cubic 
centimeters  are  measured  out  accurately  after  shaking  well,  and  they  are  made 
up  to  6400/1000  parts,  i.e.,  6.4  parts.  Multiply  6.4  x  5  and  the  result  32  equals  the 
volume  in  cubic  centimeters  to  which  the  5  c.c.  should  be  made  up.  The  suspension 
is  sterilized  by  means  of  heat.  For  staphylococci  and  streptococci  59°  to  60°  C. 
for  half  an  hour  is  sufficient;  for  typhoid  bacilli  50°  to  56°  C.  for  an  hour  is 
usually  employed.  It  is  best  to  add  some  preservative  as  phenol  or  tricresol  (0.3 
to  0.5  per  cent.)  to  the  suspension  and  to  have  the  suspension  in  sealed  ampoules 
preferably  of  brown  glass  before  immersing  in  the  water  bath.  Connor  success- 
fully sterilized  his  staphylococcic  vaccines  by  means  of  fluorides.  After  steriliza- 
tion an  ampoule  should  be  opened  so  that  a  culture  may  be  made.  No  vaccine 
should  be  used  until  a  culture  is  found  to  show  no  growth.  If  ampoules  are  not 
at  hand  they  may  be  made  from  test  tubes  or  the  vaccine  may  be  kept  in  sterile 
bottles  with  rubber  stoppers  or  caps. 

Dosage  of  Organisms. — For  gonococci,  bacillus  coli,  streptococci 
and  pneumococci  5,000,000  to  50,000,000  are  usually  employed,  while  for 
staphylococci  200,000,000  to  1,000,000,000.  Wilson  gives  the  following 
minimum  and  maximum  doses :  Streptococcus,  6  and  68  millions ; 
staphylococcus,  150  and  900  millions;  gonococcus,  45  and  900  millions; 
meningococcus,  300  and  900  millions;  micrococcus  melitensis,  700  and 
1400  millions;  bacillus  coli,  16  and  240  millions;  bacillus  typhosus  for 
treatment,  100  and  250  millions;  bacillus  typhosus  for  prophylaxis,  500 
and  1000  millions;  bacillus  pyocyaneus,  34  and  1000  millions;  bacillus 
pneumoniae,  44  millions;  bacillus  tuberculosis,  1/2000  to  1/200  mg. 

Lipovaccines. — Recently  LeMoignic  and  Sezary  showed  that  it  is 
possible  to  obtain  as  highly  hemolytic  serum  by  injecting  red  cells  sus- 
pended in  oil  as  by  injecting  them  suspended  in  salt  solution.  They  also 
showed  that  the  oil  suspension  gives  slow  absorption,  and  that  the  oil  acts 
as  a  detoxifying  agent.  As  an  example  of  the  rate  of  absorption  it  was 
shown  that  the  injection  0.35  mgm.  of  strychnine  in  aqueous  solution 
kills  a  guinea-pig,  but  the  injection  of  six  times  that  amount  is  harmless 
if  the  strychnine  be  dissolved  in  oil.  This  led  LeMoignic  and  Pinoy, 
Achard  and  Foix,  and  LeMoignic  and  Sezary  to  suspend  bacterial 
vaccines  in  oil,  and  to  inject  the  entire  vaccinating  dose  at  one  time. 
Bacterial  vaccines  suspended  in  saline  are  rapidly  autolized.  As 
autolysis  advances,  absorption  following  injection  becomes  more  rapid 
and  the  immediate  reaction  more  severe.  Oil  vaccines  are  preserved 
much  more  easily  than  saline  vaccines  and  the  reactions  following 
their  injection  are  less  severe.  The  oil  vaccines  are  known  as  "  lipo- 
vaccines."  The  bacteria  have  been  suspended  in  lanolin,  lecithin, 
sperm  oil  and  many  vegetable  oils.  Cotton-seed  oil  is  at  present  widely 
used.  It  is  of  great  importance  to  use  neutral  oils.  The  sterilization 
of  these  oils  has  been  a  difficult  problem  and  a  drawback  in  the  prepara- 
tion of  the  vaccines.  The  technic  must  be  strictly  aseptic.  At  present 
lanolin  and  oils  are  sterilized  in  the  autoclave  at  fifteen  pounds  for 


278  THE  PRINCIPLES  OF  IMMUNOLOGY 

fifteen  minutes.  Ultra-violet  rays  have  been  used.  Chlorine  has  also 
been  employed,  but  the  resulting  hydrochloric  acid  is  difficult  to  remove. 
Whitmore  and  Fennel  used  powdered  potassium  iodid.  This  was  added 
to  olive  oil  and  sweet  almond  oil;  iodin  was  liberated  in  sufficient 
amount  to  sterilize  the  oil,  and  was  taken  up  in  the  oil  molecule  so  that 
no  free  iodin  could  be  detected.  Sweet  almond  oil  is  sterilized  in  about 
three  days,  but  it  requires  about  ten  days  to  sterilize  olive  oil.  It  is  not 
known,  however,  how  the  suspension  in  oil  affects  the  antigenic  power 
of  the  vaccine,  but  certain  workers  claim  to  get  better  results  than  by 
the  use  of  saline  vaccines.  Against  the  use  of  lipovaccines  is  the 
possibility  of  fat  embolism  from  accidental  entrance  of  the  vaccine 
into  a  vein,  but  Graham  considers  this  factor  of  minor  importance  since 
the  amount  of  oil  is  small.  He  injected  as  much  as  0.8  c.c.  of  oil  into 
the  ear  vein  of  a  rabbit  and  observed  only  a  slight  passing  dyspnea 
and  no  other  evidence  of  discomfort.  Care  should  be  taken,  however, 
to  administer  the  vaccine  subcutaneously  and  to  avoid  veins.  Drawing 
out  the  plunger  of  the  syringe  after  the  needle  has  been  introduced 
determines  whether  or  not  an  important  vein  has  been  entered.  The 
upper  arm  beneath  the  insertion  of  deltoid  muscle  is  usually  selected 
for  the  injection.  The  region  of  the  scapular  or  pectoral  muscles 
may  do  as  well. 

Contraindications. — In  prophylactic  immunization  it  is  of  im- 
portance to  ascertain  whether  the  patient  has  latent  or  active 
infection.  In  active  tuberculosis  vaccination  is  considered  danger- 
ous. Caution  should  be  observed  in  diabetes,  parenchymatous  nephritis 
and  carcinoma. 

VACCINATION  WITH  LIVING  VIRUS 

Smallpox  Vaccination. — Although  inoculation  with  the  virus  in 
smallpox  in  an  attempt  to  produce  a  mild  attack  of  the  disease  had 
been  practiced  for  centuries  and  although  for  many  years  it  had  been 
observed  that  an  attack  of  cowpox  rendered  man  immune  to  smallpox, 
it  remained  for  Jenner  in  1796  to  furnish  the  scientific  proof  of  the 
efficacy  of  vaccination  with  cowpox  in  the  prevention  of  smallpox. 
Jenner's  publications  were  so  convincing  that  the  method  soon  attained 
widespread  use  and  was  introduced  into  America  in  1800  by  Dr.  Benja- 
min Waterhouse,  of  Boston.  The  work  of  the  latter  investigator  was 
especially  well  conducted  and  convincing.  In  1894  Copeman  demon- 
strated the  protection  of  monkeys  against  smallpox  by  vaccination  with 
cowpox,  and  this  was  subsequently  confirmed  by  Brinckerhoff  and 
Tyzzer.  The  introduction  of  vaccination  following  Jenner's  publication 
immediately  led  to  marked  reduction  in  the  incidence  of  this  disease 
and  its  mortality.  The  table  on  page  279  (taken  from  O'Connell,  "  Vac- 
cination; What  It  Is,  etc.,"  circular  New  York  State  Department  of 
Health,  1908)  gives  a  clear  indication  of  the  reduction  of  mortality. 

Up  until  fairly  recent  times  vaccination  was  practiced  by  inocu- 
lating patients  with  the  fragments  of  the  crust  obtained  from  others 
who  had  been  successfully  vaccinated.  This  method  has  been  aban- 


PROPHYLACTIC  VACCINATION 


279 


doned  because  of  the  possibility  of  transferring  infection  in  the  crusts, 
not  the  least  important  of  which  is  syphilis.  With  the  development  of 
important  Board  of  Health  laboratories  and  large  commercial  lab- 
oratories it  is  now  possible  to  secure  cowpox  virus  in  a  form  free 
from  infective  organisms. 

DEATH-RATE  FROM  SMALLPOX,  AVERAGE  PER  1,000,000  INHABITANTS. 


Before  vac- 
cination 

After  vaccina- 
tion 

Territory 

Smallpox  death  rate,  per  1,000,000  pop. 

Before  vaccination 

After  vaccination 

1777-1806 

1807-1850 

Austria  (lower) 

2,484 

340 

1777-1806 

1807-1850 

Austria    (upper    and 

Salzburg) 

1,421 

501 

1777-1806 

1807-1850 

Trieste 

14,046 

182 

1777-1806 

1807-1850 

Tyrol  and  Vorarlberg 

911 

170 

I777-l8o6 

1807-1850 

Bohemia 

2,174 

215 

1777-1806 

1807-1850 

Moravia 

5402 

255 

I777-l8o6 

1807-1850 

Silesia  (Austrian) 

5,812 

I98 

1776-1780 

1810-1850 

Prussia  (East   Prov- 

ince) 

3,321 

556 

1780 

1810-1850 

Prussia  (West  Prov- 

ince) 

2,272 

356 

1776-1780 

1816-1850 

Westphalia 

2,643 

114 

1776-1780 

1816-1850 

Rhenish  Provinces 

908 

90 

1781-1805 

1810-1850 

Berlin 

3,422 

I76 

I774-I80I 

1810-1850 

Sweden 

2,050 

158 

I75I-I80O 

1801-1850 

Copenhagen 

3,128 

286 

The  Preparation  of  Smallpox  Vaccine. — For  this  purpose  cow- 
pox  is  produced  in  young  heifers  from  two  to  four  months  old.  The 
animals  are  taken  from  selected  stock,  carefully  tested  for  the  presence 
of  tuberculosis  and  observed  for  several  days  so  as  to<  ensure  perfect 
health.  The  body  is  cleansed  and  the  abdominal  surface  shaved  from 
the  ensiform  cartilage  to  the  pubis,  extending  the  area  out  on  the 
flanks  and  the  inner  surface  of  the  thighs.  The  skin  is  washed  with 
soap  and  water,  then  with  alcohol  and  finally  with  sterile  water.  About 
100  small  cuts  through  the  epidermis  are  made  under  strictly  aseptic 
precautions.  If  bleeding  occurs  the  blood  is  carefully  wiped  away. 
Virus  may  be  obtained  primarily  from  smallpox  patients  who  are 
otherwise  healthy.  At  the  present  time,  however,  the  virus  kept  as 
"  seed  virus  "  is  obtained  from  previously  inoculated  animals.  Virus 
is  introduced  into  the  scarifications  usually  in  the  form  of  a  glycerol 
suspension.  In  about  forty-eight  hours  a  reaction  appears  and  by  the 
sixth  day  the  vesicles  are  well  filled  with  semipurulent  material.  The 
animal  is  killed  and  the  vesicles  carefully  curetted  away.  After  the 
curettage,  serum  appears  and  this  may  be  preserved  in  ampoules  or 
small  tubes  for  subsequent  vaccination.  The  pulpy  mass  obtained 
by  curettage  is  mixed  with  four  times  its  weight  of  a  mixture  com- 
posed of  glycerol  50  per  cent.,  water  49  per  cent.,  phenol  i  per  cent. 
The  glycerolated  pulp  is  allowed  to  stand  three  or  four  weeks  in  order 
to  destroy  any  contaminating  bacteria.  The  pulp  is  then  triturated  in 


280  THE  PRINCIPLES  OF  IMMUNOLOGY 

special  machines  and  sealed  in  capillary  tubes.  Formerly  "  ivory " 
vaccine  points  were  also  charged  from  this  pulp,  but  these  have  been 
forbidden  in  interstate  commerce  (page  282).  In  all  cases  the  material 
before  being  prepared  for  distribution  is  carefully  tested  for  the 
presence  of  tetanus  bacilli  or  their  spores.  Its  potency  may  be  deter- 
mined by  directly  inoculating  the  inner  surface  of  the  ears  of  rabbits 
and  observing  the  rapidity  of  the  reaction.  A  somewhat  superior 
method  is  to  make  dilutions  of  the  virus  and  to  note  the  effect  of  these 
dilutions  when  inoculated  on  the  ears  of  rabbits.  A  potent  virus  should 
produce  vesicles  in  a  dilution  of  i  to  500.  Efforts  have  been  made  to 
secure  a  virus  in  purer  form  and  Noguchi  has  planted  the  virus  in  the 
testicles  of  rabbits  and  of  bulls.  Virus  recovered  from  this  situation 
is  not  subject  to  contamination  in  the  same  way  as  that  obtained  from 
surface  inoculations.  The  amount  of  material  obtained,  however,  is 
small  and  the  method  has  not  been  used  extensively  enough  to  justify 
an  opinion  as  to  its  value.  After  preparation  of  a  virus  the  date 
should  be  indicated  on  the  container  and  the  material  preserved  in 
the  ice  chest. 

Methods  of  Inoculation  in  Man. — As  a  rule,  vaccination  is  applied 
on  the  upper  arm  over  the  point  of  insertion  of  the  deltoid  muscle. 
This  situation  offers  protection  against  injury  and  contamination  such 
as  is  not  afforded  by  vaccination  upon  the  leg  or  thigh.  The  area  is 
carefully  cleansed  with  soap  and  water,  followed  by  alcohol  or  ether 
and  then  by  distilled  water.  The  last  step  is  sometimes  omitted.  For- 
merly the  area  was  scarified  in  a  criss-cross  manner  by  means  of  a 
needle  or  scalpel,  but  such  extensive  scarification  has  been  found  to 
be  unnecessary  and  also  exposes  a  greater  surface  to  the  possibility 
of  infection.  The  more  modern  method  is  to  place  the  virus  upon  the 
area  and  to  make  a  scarification  through  the  virus.  This  may  be  done 
by  a  small  linear  incision,  by  the  drill  method  or  by  the  multiple  punc- 
ture method.  Wright  has  advised  intracutaneous  inoculation. 

Method  of  Linear  Incision. — After  placing  the  virus  upon  the  skin 
a  sterile  needle  or  a  small  scalpel  is  employed  for  making  a  scarification 
through  the  virus  and  sufficiently  deep  into  the  skin  to  permit  absorption 
but  not  to  produce  bleeding.  The  virus  is  then  gently  rubbed  into  the 
abrasion  and  permitted  to  dry.  If  a  dressing  is  desired  it  should  be  of 
sterile  gauze  loosely  applied  with  adhesive  strips  after  the  virus  has 
completely  dried.  Sealing  with  collodion  should  not  be  attempted, 
since  it  may  permit  more  ready  growth  of  contaminating  bacteria 
and  produce  maceration  of  the  skin. 

The  Drill  Method.— A  sterile  drill  such  as  is  employed  in  the  Von 
Pirquet  cutaneous  tuberculin  test  is  held  between  the  thumb  and  middle 
finger.  With  a  twisting  motion  and  moderately  firm  pressure  a  small 
abrasion  the  diameter  of  the  drill  is  made  through  the  virus.  This 
should  penetrate  the  epiderm,  but  should  draw  no  blood. 

The  Multiple  Puncture  Method. — A  sterile  needle  is  held  nearly 
parallel  with  the  skin  and  the  point  placed  through  a  drop  of  virus  so  as 


PROPHYLACTIC  VACCINATION  281 

to  make  an  oblique  puncture  of  the  epidermis.  This  is  repeated  so  as 
to  produce  about  six  radially  disposed  punctures,  the  whole  area  ex- 
tending not  more  than  about  5  mm. 

The  Intracutaneous  Method. — The  virus  is  diluted  to  ten  times  its 
volume  with  distilled  water  and  injected  intracutaneously  by  means 
of  a  sterile  tuberculin  syringe  and  a  fine  needle.  Two  injections  about 
2  cm.  apart  are  made. 

All  the  methods  indicated  have  given  equally  good  results,  but 
convenience  usually  dictates  the  use  of  the  linear  incision  or  the  drill 
method.  It  is  not  uncommon  in  the  use  of  any  of  these  methods  to 
make  two  or  three  inoculations. 

Vaccinia. — Following  the  inoculation  of  the  virus  the  areas  usually 
remain  quiescent  for  from  two  to  four  days  when  slight  reddening  and 
itching  may  develop.  Following  this  a  small  papule  appears,  rapidly 
succeeded  by  the  vesicle.  It  is  important  to  note  that  the  vesicle  is 
umbilicated  and  that  its  multilocular  character  is  indicated  by  the 
minute  vesicular  arrangement  of  the  margin.  The  vesicle  appears  in 
from  five  to  six  days,  rapidly  becomes  pustular  and  is  followed  by  the 
formation  of  the  crust.  The  crust  is  allowed  to  drop  off  and  subse- 
quent observations  of  the  scar  should  show  a  smooth  center,  a  somewhat 
scalloped  edge  and  more  or  less  discrete  minute  marginal  scars.  During 
the  height  of  the  local  reaction  the  patient  may  complain  of  malaise, 
headache,  fever,  constipation  and  other  general  symptoms.  The  reac- 
tion of  vaccinoid  has  been  discussed  in  the  chapter  on  Hypersuscep- 
tibility  (page  243). 

Immunity  as  the  Result  of  Vaccination. — The  extent  of  immunity 
has  been  indicated  by  the  decrease  in  prevalence  of  smallpox  since  the 
introduction  of  vaccination.  It  may  also  be  measured  by  the  success 
of  subsequent  vaccinations.  Kitasato  has  revaccinated  a  series  of  931 
cases  with  successful  results  as  follows : 

After  i  year  14  per  cent.  After  6  years,64  per  cent. 

After  2  years  33  per  cent.  After  7  years  73  per  cent. 

After  3  years  47  per  cent.  After  8  years  80  per  cent. 

After  4  years  57  per  cent.  After  9  years  85  per  cent 

After  5  years  51  per  cent.  After  10  years  89  per  cent. 

It  will  thus  be  seen  that  more  than  50  per  cent,  of  individuals  are 
susceptible  to  revaccination  four  years  after  the  original  vaccination. 
Millard  states  that  the  Government  reports  of  the  German  Confederacy 
show  91  per  cent,  to  93  per  cent,  successful  revaccinations  in  ten  years 
or  more  after  the  primary  vaccination  and  concludes  that  "  immunity 
acquired  through  vaccination  begins  to  disappear  at  about  the  second 
year  and  by  the  tenth  year  it  disappears  almost  completely."  Other 
investigators  have  obtained  similar  results.  King  reported  that  in 
ninety-six  adults  who  had  suffered  from  smallpox  at  various  ages  and 
showed  numerous  scars  of  the  disease,  vaccination  was  successful  in 
75  per  cent.  These  figures  indicate  that  the  older  conceptions  of  the 


282  THE  PRINCIPLES  OF  IMMUNOLOGY 

durability  of  the  immunity  produced  by  vaccination  are  inaccurate. 
In  order  to  secure  satisfactory  immunity,  vaccination  should  be  re- 
peated at  intervals  of  a  few  years.  In  those  communities  where  small- 
pox is  endemic  vaccination  should  be  repeated  every  year.  In  the 
presence  of  epidemics,  an  unsuccessful  vaccination  should  not  be  inter- 
preted as  indicating  immunity  and  should  be  repeated  at  intervals  of  a 
week  or  ten  days  until  successful.  We  feel  that  no  dependence  can 
be  unqualifiedly  placed  on  the  signs  of  immunity  as  indicated  by 
Force  (page  243). 

Unfavorable  Results  of  Vaccination. — If  human  virus  be  employed 
the  chance  of  inoculating  syphilis  must  be  considered,  although  the 
danger  is  slight.  Reports  of  tetanus  following  shortly  after  vaccina- 
tion have  not  been  particularly  well  founded  and  examination  of  a 
large  number  of  samples  collected  by  the  Hygienic  Laboratory  in 
Washington  by  McCoy  and  Bengston  failed  to  demonstrate  the  pres- 
ence of  the  bacilli  or  their  spores  in  filled  capillary  tubes,  seed  vaccine 
or  in  bulk  glycerolated  vaccine.  "  Ivory  points  "  were  found  to  be 
contaminated  as  delivered  from  the  manufacturer  of  the  points,  as  well 
as  after  sterilization  and  charging.  McCoy  states  that  "  the  sale  of 
vaccine  virus  on  or  with  points  in  interstate  traffic  has  been  prohibited 
by  an  order  of  the  Secretary  of  the  Treasury.'* 

The  most  important  source  of  trouble  is  the  result  of  vaccination  in 
unclean  skin,  the  use  of  unclean  dressings  or  other  failures  of  asepsis, 
more  particularly  those  resulting  from  carelessness  on  the  part  of  the 
patient.  Such  infections  usually  remain  localized  but  confuse  the 
interpretation  of  results  and  may  in  rare  instances  become  gen- 
eral infections. 

Vaccination  Against  Rabies. — The  cause  of  rabies  is  probably  a 
sporozoan  parasite  discovered  by  Negri  and  named  by  Calkins  "  neuro- 
ryctes  hydrophobise."  Work  with  this  parasite  is  difficult  because  of 
failure  to  isolate  the  organism  in  suitable  form.  Therefore,  the  investi- 
gations have  been  conducted  with  pathological  material  containing  the 
organism.  It  is  found  in  greatest  amounts  in  the  nervous  system  and 
accordingly  the  brain  or  cord  is  selected  for  experimental  work.  This 
material  is  spoken  o<f  as  the  virus  of  rabies.  Street  virus  is  nerve 
tissue  obtained  from  an  animal  suffering  with  the  natural  disease.  It 
is  extremely  variable  in  virulence,  and  for  this  reason  is  not  employed 
for  vaccination  of  man.  Fixed  virus  is  usually  the  spinal  cord  of 
rabbits  obtained  after  a  long  series  of  rabbit  passages.  By  these  animal 
passages  the  virulence  increases  and  the  incubation  period  decreases 
until  a  point  is  reached  when  the  incubation  period  following  inocula- 
tion cannot  be  further  shortened.  All  mammals  are  susceptible  to  rabies 
in  different  degrees,  but  birds  or  reptiles  are  not  susceptible. 

The  treatment  of  rabies  in  man  after  it  has  developed  has  been 
entirely  unsatisfactory  by  the  methods  of  immunology.  Immunization 
of  animals  to  the  rabies  virus  produces  an  immune  serum  capable  of 
killing  the  virus.  Accordingly  it  was  hoped  that  such  a  serum  could 


PROPHYLACTIC  VACCINATION  283 

be  employed  for  human  rabies,  but  results  attendant  upon  this  method 
of  treatment  have  been  unsuccessful.  Therefore,  at  the  present  time, 
efforts  are  directed  toward  producing  an  active  immunity  in  those  who 
have  been  exposed  to  the  disease.  It  is  of  interest  to  note  that  laboratory 
inoculations  in  man  rarely,  if  ever,  lead  to  the  development  of  the 
disease.  It  is  probable  that  in  order  for  infection  to  occur  the  virus 
must  be  implanted  with  animal  sputum  or  some  other  form  of  con- 
tamination. Bites,  from  rabid  dogs  are  relatively  infrequent,  and  it  is 
therefore  unnecessary  to  immunize  an  entire  population.  Further- 
more, man  is  somewhat  resistant  to  infection  with  rabies.  Statistical 
evidence  in  regard  to  the  frequency  with  which  rabies  follows  the  bites 
of  rabid  dogs  are  unreliable  because  of  uncertainty  as  to  whether  or 
not  the  animal  was  rabid.  Doebert  found  that  in  Prussia,  where  data 
had  been  very  carefully  collected,  there  was  a  mortality  of  14.8  per 
cent,  in  122  untreated  persons  bitten  by  rabid  animals  between  the  years 
1902  and  1907.  Other  estimates  conform  closely  to  this.  More  recently, 
however,  Marx  has  expressed  the  opinion  that  the  rate  of  mortality 
probably  does  not  exceed  6  per  cent,  to  10  per  cent,  of  untreated 
bitten  persons.  The  mortality  and  morbidity  rate  are  practically  identical. 
Fortunately  the  period  of  incubation  of  rabies  is  of  sufficiently  long 
duration  so  that  active  immunization  may  be  effected  during  the  period 
of  incubation. 

The  period  of  incubation  in  man  is  variable  and  depends  to  a  con- 
siderable extent  upon  the  site  of  the  bite  or  scratch.  According  to 
Bauer,  the  average  period  of  incubation  in  510  cases  was  seventy-two 
days.  In  very  rare  cases  the  period  of  incubation  may  be  less  than 
nineteen  days  and  in  more  rare  instances  it  may  be  one  year  or  more. 
Of  seventy- three  cases  of  bites  about  the  head  and  neck  the  average 
incubation  was  fifty-five  days;  of  144  cases  of  bites  on  the  upper 
extremities  the  average  period  was  eighty-one  and  one-half  days ; 
and  of  seventeen  cases  of  bites  on  the  lower  extremities  the  average 
period  of  incubation  was  seventy-four  days. 

Active  Immunization.  Preparation  of  Material. — As  has  been 
indicated  above  it  is  necessary,  because  of  the  failure  of  passive 
immunization,  to  produce  an  active  immunization.  In  spite  of  the  fact 
that  laboratory  accidents  practically  never  lead  to  the  development  of 
rabies  it  is  considered  dangerous  to  inoculate  man  with  the  living  virus. 
Ferran  and  subsequently  Proescher  have,  however,  employed  a  method 
whereby  the  active  fixed  virus  is  employed.  Both  these  investigators 
stated  that  no  accidents  had  followed  the  use  of  unmodified  fixed  virus. 
Hogyes  has  successfully  employed  dilutions  of  fresh  fixed  virus.  The 
majority  of  investigators,  however,  have  employed  virus  which  has 
been  attenuated  by  a  variety  of  methods  including  heat,  partial  diges- 
tion, the  action  of  bile,  the  action  of  glycerol,  of  anti-rabic  serum,  of 
phenol  and  of  mechanical  disintegration.  Nevertheless,  the  original 
method  of  Pasteur  is  employed  almost  uniformly  throughout  the  world. 
For  this  purpose  the  virus  is  passed  through  rabbits  until  it  acquires 


284  THE  PRINCIPLES  OF  IMMUNOLOGY 

its  minimum  period  of  incubation.  The  material  is  introduced  into  the 
anesthetized  rabbit  by  subdural  inoculation.  The  injection  is  made 
through  a  small  trephine  opening  just  back  of  the  eye  and  to  one  side 
of  the  median  line.  The  injected  material  is  ground  with  a  small 
quantity  of  I  per  cent,  phenol  solution  and  0.2  c.c.  of  this  emulsion  is 
injected.  After  the  rabbit  is  completely  paralyzed  it  is  killed  with 
chloroform  and  the  spinal  cord  removed  aseptically.  A  small  ligature 
is  placed  around  one  end  of  the  cord  and  the  cord  hung  in  a  sterile 
bottle  in  the  bottom  of  which  has  been  placed  sticks  of  potassium 
hydrate.  The  bottle  is  placed  in  an  incubator  maintained  at  22°  to  23°  C. 
Pieces  I.  cm.  in  length  are  cut  off  at  daily  intervals  and  placed  in  glycerol 
where  the  degree  of  virulence  on  that  particular  day  is  retained  for 
several  weeks.  In  large  laboratories  animals  may  be  killed  on  suc- 
cessive days  and  the  whole  cord  employed  in  preparing  the  material 
for  human  protection.  In  the  United  States  Hygienic  Laboratory 
pieces  0.5  cm.  in  length  emulsified  with  2.5  c.c.  of  salt  solution  serve 
for  one  injection. 

Inoculation  in  Man. — The  determination  as  to  who  shall  receive 
anti-rabic  treatment  is  often  difficult,  but  skilled  veterinarians  are 
able  to  diagnose  rabies  in  dogs  almost  invariably.  Knowledge  of  the 
condition  of  the  animal  inflicting  a  bite  is  of  the  utmost  importance. 
Although  cats  and  rats  are  not  uncommonly  victims  of  rabies,  this  is 
not  frequently  a  source  of  infection  in  man.  When  a  dog  bite  is  re- 
ceived, the  animal  should  be  captured  and  observed  for  at  least  two 
weeks,  during  which  time  the  symptoms  of  rabies  become  manifest. 
If  the  animal  is  killed  the  brain  should  be  sent  in  glycerol  to  the  nearest 
laboratory,  where  it  may  be  examined  for  Negri  bodies.  If  these  are 
not  found,  material  should  be  injected  into  rabbits.  Negative  findings 
in  regard  to  Negri  bodies  in  the  dog's  brain  are  not  to  be  accepted  as 
evidence.  In  our  opinion  it  is  wise  to  administer  treatment  to  all 
individuals  who  have  been  bitten  by  animals  showing  any  signs  of 
rabies.  The  material  may  be  supplied  to  the  physician  either  in  the 
form  of  small  pieces  of  cord  to  be  emulsified  in  salt  solution  or  in  the 
form  of  an  emulsion  for  dilution  with  salt  solution.  The  injections 
are  given  subcutaneously  under  the  skin  of  the  abdomen.  If  a  con- 
siderable time  has  elapsed  since  the  bite  or  if  the  bite  has  been  inflicted 
upon  the  head  or  neck  the  so-called  intensive  method  of  treatment  is 
adopted.  Under  other  circumstances  the  mild  treatment  may  be  given. 
When  material  is  requested  from  a  commercial  laboratory  or  a  state 
laboratory  it  is  necessary  to  indicate  which  form  of  treatment  is  desired. 
As  an  example  of  the  two  methods,  the  scheme  of  treatment  as  shown  on 
page  285,  adopted  by  the  New  York  City  Board  of  Health,  will  serve. 

The  Effects  of  Treatment.— Local  reactions  are  frequent  and  are 
likely  to  be  severe  about  the  eleventh  and  nineteenth  days  of  inocula- 
tion. These  are  urticarial  in  character  and  the  more  severe  reactions 
may  be  accompanied  by  mild  constitutional  symptoms.  The  glycerol 
contained  in  the  emulsions  not  infrequently  produces  severe  pain  for 


PROPHYLACTIC  VACCINATION  285 

a  few  moments  at  the  site  of  inoculation.  The  treatment,  although 
practically  safe,  is  not  entirely  free  from  danger.  Remlinger  in  a 
study  of  107,712  cases  that  had  received  treatment  found  forty  cases 
which  developed  paralysis  of  the  extremities  and  two  of  these  ter- 
minated in  death.  The  cause  of  this  paralysis  is  not  clear.  Certain 
authorities  maintain  that  the  virus  contains  a  toxin  and  that  this  may 
lead  to  lesions  of  the  nerves.  The  mass  of  evidence,  however,  is 
against  rather  than  in  favor  of  the  conception  that  toxin  plays  any 
important  part  in  the  virus  of  rabies.  It  is  also  possible  that  the 
repeated  injections  of  foreign  protein  may  have  some  influence.  Such 
accidents  are  extremely  rare  and  should  not  interfere  with  a  decision 
concerning  administration  of  the  treatment. 

SCHEME  OF  TREATMENTS. 
Day  Mild  treatment  Intensive  treatment 

ist  14  and  13  day  cord  12  and  n  day  cord,  repeat 

in  afternoon 

2nd  12  and  u  day  cord  10  and  9  day  cord;  8  and  7 

day  cord  in  afternoon 

3rd  10  and     9  day  cord  6  day  cord 

4th  8  and     7  day  cord  5  day  cord 

5th  6  day  cord  4  day  cord 

6th  5  day  cord  3  day  cord 

7th  4  day  cord  2  day  cord 

8th  3  day  cord  4  day  cord 

9th  2  day  cord  4  day  cord 

loth  4  day  cord  i  day  cord 

nth  3  day  cord  4  day  cord 

I2th  2  day  cord  3  day  cord 

I3th  4  day  cord  2  day  cord 

I4th  5  day  cord  4  day  cord 

I5th  2  day  cord  I  day  cord 

i6th  4  day  cord  4  day  cord 

I7th  3  day  cord  3  day  cord 

i8th  2  day  cord  2  day  cord 

I9th  4  day  cord  4  day  cord 

2oth  3  day  cord  3  day  cord 

2ist  2  day  cord  2  day  cord 

Results  of  Treatment. — The  benefits  of  this  form  of  treatment 
depend  to  a  certain  extent  upon  the  time  when  the  injections  are  begun 
and  also  to  a  certain  extent  upon  the  situation  of  the  bite.  Granting 
that  fatalities  occur  in  from  6  to  16  per  cent,  of  untreated  bitten  indi- 
viduals, the  reports  of  fatalities  in  from  .46  per  cent,  to  1.25  per  cent, 
of  treated  cases  show  markedly  beneficial  effects.  More  recent  statistics 
are  highly  encouraging.  During  the  year  1916  Viala  reported  that  654 
persons  were  treated  at  the  Pasteur  Institute  with  but  one  death. 

VACCINATION  WITH  KILLED  ORGANISMS 

Vaccination  Against  Typhoid  and  Paratyphoid  Fevers. — Al- 
though various  investigators  had  appreciated  the  possibility  of  active 
immunization  against  typhoid  fever,  this  subject  was  first  placed  on  a 
practical  basis  by  Wright  in  1896.  In  the  subsequent  year  Wright 
and  Semple  described  in  detail  a  satisfactory  method  for  vaccination. 


286  THE  PRINCIPLES  OF  IMMUNOLOGY 

They  employed  broth  cultures  of  bacillus  typhosus  two  to  three  weeks 
old,  killed  by  heating  to  63°  C.  for  one  hour  and  preserved  with  0.5 
per  cent,  phenol.  The  vaccine  was  treated  for  sterility,  standardized 
and  employed  in  doses  of  750  to  1000  million  organisms.  In  the  same 
year  Pfeiffer  and  Kolle  reported  the  demonstration  of  specific  anti- 
bodies following  the  immunization  of  man  against  the  organism.  Since 
that  time  vaccines  have  been  prepared  in  a  large  variety  of  ways  and 
preventative  vaccination  is  now  upon  a  highly  satisfactory  basis.  Vac- 
cination has  been  employed  in  military  and  civil  life  and  has  resulted 
in  a  marked  decrease  in  morbidity  and  mortality.  The  results  obtained 
in  all  civilized  countries  constitutes  one  of  the  greatest  achievements 
resulting  from  the  study  of  immunology. 

Preparation  of  Vaccines. — The  organisms  may  be  grown  in  broth  or 
upon  agar.  The  broth  culture  or  a  salt  solution  suspension  of  an  agar 
culture  may  be  killed  or  attenuated.  The  application  o>f  heat  or  chemi- 
cals for  the  purpose  of  killing  the  organisms  reduces  in  a  certain 
measure  their  antigenic  value.  If  they  are  dried  before  being  heated, 
temperatures  of  120°  to  150°  C.  reduce  the  antigenic  property  very 
little.  Gay,  however,  points  out  that  the  measure  of  the  antigenic  value 
depends  upon  the  determination  of  different  antibodies  such  as  agglu- 
tinins  and  bacteriolysins,  but  he  notes  that  this  offers  "an  indication 
rather  of  the  reaction  of  the  animal  body  than  a  sure  means  of  deter- 
mining the  degree  of  protection  that  has  actually  been  afforded." 
Numerous  investigators  have  suggested  the  use  of  living  bacteria,  but 
the  knowledge  that  typhoid  fever  may  exist  as  a  septicemia  without 
intestinal  lesions  offers  an  objection,  to  the  introduction  of  living  or- 
ganisms. It  has  been  found  extremely  difficult  to  attenuate  without 
killing  the  bacteria,  but  it  has  been  recommended  that  low  degrees  o>f 
temperature,  for  example  53°  C.  (Leishman),  the  use  of  ether,  alcohol, 
various  sugars  and  other  chemical  and  physical  agents  may  kill  the 
organisms  without  markedly  reducing  the  antigenic  properties.  Certain 
investigators  have  also  suggested  the  employment  of  bacterial  extracts, 
but  this  method  has  not  been  widely  employed.  Gay  and  his  collab- 
orators (page  301)  have  claimed  success  in  the  therapeusis  of  typhoid 
fever  by  the  use  of  sensitized  vaccines  and  have  found  that  active 
immunization  progresses  very  satisfactorily,  according  to  measurements 
of  specific  antibodies,  yet  this  method,  if  employed  for  vaccination, 
is  expensive  and  probably  does  not  give  sufficiently  superior  results  to 
justify  its  employment  in  large  numbers  of  individuals, 

It  is  now  recognized  that  the  typhoid  bacillus  may  be  divided  into 
a  number  of  strains  on  the  basis  of  cultural  and  immunological  prop- 
erties. In  certain  countries,  including  the  United  States,  a  single  strain 
of  the  organism  has  been  employed  for  vaccination,  but  in  others  a  poly- 
valent vaccine  has  been  employed,  the  French  using  ten  strains,  The 
organisms  are  grown  on  large  agar  surfaces,  emulsified  in  salt  solution 
and  killed  by  heat.  They  are  then  standardized  and  a  preservative, 
such  as  phenol,  lysol  or  formaldehyde,  added.  Twenty-four  hours  sub- 


PROPHYLACTIC  VACCINATION  287 

sequently  cultures  are  made  to  determine  the  sterility  of  the  vaccine. 
Standardization  is  usually  on  the  basis  of  1000  million  organisms  per 
cubic  centimeter.  If  it  be  desired  to  give  a  smaller  number  of  organisms, 
fractions  of  a  cubic  centimeter  may  be  employed.  It  is  the  practice  in 
commercial  houses  to  place  specified  doses  in  small  ampoules  so  that 
the  physician  may  administer  for  each  dose  the  contents  of  a  single 
ampoule.  In  military  practice  the  vaccine  is  placed  in  small  bottles  with 
a  rubber  cap  so  that  a  needle  may  be  thrust  through  the  cap  and  the 
required  amount  of  vaccine  withdrawn  into  a  syringe. 

As  the  paratyphoid  fevers  have  been  studied,  it  has  been  considered 
advisable  to  vaccinate  against  these  at  the  same  time  as  against  typhoid 
fever.  Therefore,  vaccines  are  now  prepared  containing  the  bacillus 
typhosus,  bacillus  paratyphosus  A  and  bacillus  paratyphosus  B.  It  has 
been  customary  to  introduce  smaller  quantities  of  the  paratyphoid 
bacilli  so  as  not  to  increase  to  an  unfavorable  degree  the  bulk  of  for- 
eign protein  injected.  Accordingly  for  each  1000  million  typhoid  bacilli 
there  are  usually  added  500  million  each  of  paratyphoid  A  and  B.  The 
actual  numbers,  however,  vary  in  different  countries.  Castellani  rec- 
ommends the  addition  also  of  cholera  vibrios.  This  transforms  the 
triple  vaccine  into  a  tetra  vaccine.  In  northern  latitudes  this  is  not  of 
particular  importance. 

As  has  been  indicated,  the  organisms  are  usually  suspended  in  salt 
solution,  but  recently  neutral  oil,  such  as  commercial  cottonseed  oil, 
has  been  employed  for  suspension.  For  such  suspension  the  organisms 
must  be  very  carefully  dried  before  being  emulsified  in  the  oil.  These 
lipovaccines  have  the  advantage  of  being  administered  in  one  dose  and 
of  producing  little  or  no  reaction.  They  produce  immunity  following 
a  single  injection  because  of  the  slow  absorption  of  the  oil  and  its 
contained  antigen. 

Method  of  Administration. — In  the  case  of  the  lipovaccines  a  single 
large  dose  of  organisms  may  be  administered.  The  use  of  the  salt 
solution  suspensions  involves  several  injections.  As  a  rule,  the  first 
dose  contains  500  million  typhoid  bacilli  and  250  million  each  of  para- 
typhosus A  and  B.  The  second  and  third  doses  contain  1000  million 
typhoid  bacilli  and  500  million  each  of  the  paratyphoid  bacilli.  The 
time  between  injections  has  been  the  subject  of  considerable  study,  but, 
as  a  rule,  a  period  of  seven  to  ten  days  intervenes  between  these  injec- 
tions. Subcutaneous  administration  is  practically  universal.  Intra- 
venous injections  have  been  recommended,  but  this  method  is  not  widely 
practiced.  Lumiere  and  Chevrotier  have  administered  by  mouth  gela- 
tine-coated pills  of  a  dried  mixed  polyvalent  typhoid  colon  vaccine.  It 
is  probable  that  this  method  is  not  effectual,  since  the  bacterial  protein 
must  undergo  at  least  partial  digestion  in  the  intestinal  tract.  Bes- 
redka,  however,  has  recently  demonstrated  in  animals  the  possibility 
of  successful  vaccination  through  the  intestinal  tract,  but  his  animals 
had  previously  been  given  bile,  and  it  seems  likely  that  this  substance 
produced  sufficient  lesion  of  the  intestinal  mucosa  to  permit  of 
direct  absorption. 


288 


THE  PRINCIPLES  OF  IMMUNOLOGY 


Prophylactic  Value  of  Vaccination. — It  can  readily  be  understood 
that  the  control  of  individuals  in  armies  offers  excellent  facilities  for 
determination  of  the  prevalence  and  mortality  of  infectious  disease. 
Consequently,  much  of  the  statistical  evidence  favorable  to  typhoid 
vaccination  has  been  collected  in  armies.  The  following  table,  taken 
from  Gay,  "Typhoid  Fever,"  illustrates  the  prevalence  of  typhoid  fever 
in  Great  Britain  and  her  colonies  before  vaccination  was  introduced : 

MORBIDITY  AND  MORTALITY  FIGURES  FROM  TYPHOID  FEVER  PER  100,000  OF  ENGLISH 
TROOPS  IN  THE  YEAR  1898  IN  DIFFERENT  LOCALITIES. 

Locality  Morbidity  Mortality 

Great  Britain  120  24 


Gibraltar    420 

South  Africa  3290 

India   3600 

Egypt    8100 


132 

577 

IOOO 

2340 


Gay  states  that  even  greater  rates  of  typhoid  morbidity  have  been  en- 
countered. The  results  of  anti-typhoid  vaccination  are  splendidly 
summarized  in  another  table  from  Gay's  work : 


RESULTS  OF  ANTI-TYPHOID  IMMUNIZATION  IN  THE  ENGLISH  ARMY. 
AND  MORTALITY  PER  100,000. 


MORBIDITY 


Locality 

Vaccinated 

Unvaccinated 

Authority 

No. 

Morbid. 

Mortal. 

No. 

Morbid. 

Mortal. 

India  1900  

I050I 

5473 
58481 

10378 

914 
380 
260 

539 

161 
36 
29 

40 

83135 
6610 
10794 

8936 

I665 
2830 
1390 

3040 

444 
390 
220 

500 

Wright. 
Leishmann. 
Firth. 

Report   of   the 
anti-  typhoid 
committee, 
London,  1913 

India  1909  
India  1910  
Various  colonies 

IQI7.  . 

The  results  obtained  in  the  United  States  Army  under  the  direction 
of  Colonel  Russell  and  his  staff  have  been  most  impressive.  In  Decem- 
ber of  1919  Colonel  Russell  summarized  the  results  in  a  paper  in  the 
Journal  of  the  American  Medical  Association.  He  gives  an  analysis 
of  a  water-borne  epidemic  in  Hawaii  as  follows : 

TYPHOID  EPIDEMIC  IN  HAWAII,  H.  T.,  IN  THE  FALL  OF  1917. 


Population  No.  of 

on  Castner  cases 

water  of 

system  typhoid 

Vaccinated   4087  55 

Unvaccinated    812  45 


Cases 

per 
thousand 

1345 

55-41 


Mortality 

Deaths  rate 

Number     Per  cent.  per 

thousand 


74 

15-5 


o.97 
8.62 


It  is  of  importance  to  note  in  reading  this  table  the  large  number 
of  vaccinated  as  contrasted  with  the  Unvaccinated.  It  is  apparent  that 
the  vaccinated  show  not  only  a  reduced  morbidity  percentage,  but  also 
a  diminished  mortality  rate.  Colonel  Russell  gives  the  following  table 
of  figures  from  the  United  States  Army  for  nineteen  years : 


PROPHYLACTIC  VACCINATION 


289 


RATE  OF  TYPHOID  FEVER  IN  THE  ARMY  AND  IN  THE  CORRESPONDING  AGE  GROUP 
IN  CIVIL  LIFE  FOR  THE  PAST  EIGHTEEN  YEARS. 


Year 

No.  of  cases 

Ratio  per 
thousand 

Deaths 

Ratio  per 
thousand 

Total 

Males* 

IQOO  
IQOI  
1902  

IQOt 

531 

594 
565 

^48 

575 
943 

8.58 
5.82 

60 
78 
69 
3° 

0-43 
0.64 
0.86 
0.28 

0.46 
0.42 
0.40 
0.35 

0-54 

IQO4.  .  . 

247 

5.62 

12 

0.27 

0.33 

1905  
IQO6.  . 

193 
747 

3-57 
5.66 

17 

15 

0.30 
0.28 

0.32 
0.32 

IQO7.  . 

208 

3-53 

16 

0.19 

0.28 

1908  
IQOQ 

215 

177 

2.94 
^..OT, 

21 

16 

0.23 
0.28 

0.28 
0.23 

I9IO  

I9nf  
1912  

IQIV  • 

142 

44 

18 
4 

2.32 
0.85 
0.31 
0.04 

IO 

6 

3 
o 

0.16 
0.09 
0.04 
o.oo 

0.27 
0.23 

0.18 
0.18 

0-34 

I9H  
1915  
IQl6 

8 

2S 

0.07 
0.08 
0.2^ 

3 
o 

7 

0.03 

0.00 

0.03 

0.15 
0.18 

O.I2 

0.17 
O.I  5 

IQI7 

2Q7 

0.44 

2^ 

O.OT, 

O.II 

O.I4 

1918  

768 

0.30 

133 

0.05 

O.09 

O.II 

*  Indicates  voluntary  vaccination  against  typhoid. 

t  Indicates  compulsory  vaccination  against  typhoid. 

t  Civil  deaths  from  typhoid  fever;  age  group,  20  to  29  years.    Rate  per  thousand  of  population. 

The  marked  change  after  the  introduction  of  compulsory  vaccination 
in  the  Army  in  1911  is  most  striking.  It  is  pointed  out  that  the  increase 
in  1917  is  in  large  part  contributed  to  by  delay  in  the  vaccination  and 
sanitary  control  of  National  Guardsmen.  As  an  impressive  contrast 
the  following  table  illustrates  the  vast  improvement  in  health  condi- 
tions as  compared  with  previous  wars : 

RELATION  OF  MORTALITY  IN  THE  WORLD  WAR  TO  THAT  OF  PREVIOUS  WARS. 


Number  of  deaths 

that  occurred  in 
the  World  War, 
Sept.  I,  1917- 
May  2,  1919. 
Average  strength 
appro  ximately 

Number  of  deaths 
that  would  have 
occurred  if  the 
Civil  War  death 
rate  had  obtained 

Number  of  deaths 
that  would  have 
occurred  if  the 
Spanish-American 
War  death  rate 
had  obtained 

2,121,396 

Typhoid  fever  

213 

51,133 

68,164 

Ma.ls.ria 

t* 

17  QCI* 

II  ^17 

Dysentery        •  • 

xo 

42 

OjVO  •*• 

63,8o8t 

*  "MO^-1/ 

6i82t 

L^t 

^J\jy^y^J  ' 

\J)£\j£t  1 

*  Includes  malaria,  remittent  and  congestive  fevers, 
t  Includes  dysentery  and  diarrhea. 

During  the  period  of  the  American  participation  in  the  World  War 
there  were  1065  cases  of  typhoid  fever  in  approximately  4,000,000 
troops,  a  ratio  of  one  case  to  every  3756  men.  In  the  Spanish- American 
War  there  was  one  case  to  every  seven  men.  Colonel  Russell's  final 
comment  is  of  the  greatest  interest.  "It  is  evident  from  these  tables, 
therefore,  that  anti-typhoid  vaccination,  carried  out  as  it  was  by  a  per- 
sonnel which  had  not  been  carefully  trained  in  its  administration,  gave 
a  high  degree  of  protection  to  our  forces  under  the  conditions  of  hur- 
ried mobilization  and  of  warfare,  and  reduced  the  rate,  not  only  below 
19 


290  THE  PRINCIPLES  OF  IMMUNOLOGY 

the  rates  for  previous  wars,  but  also  below  the  rate  found  in  civil  life  in 
some  of  the  older  states  where  the  entire  population  is  protected  by  all 
the  sanitary  measures  of  modern  life." 

At  the  beginning  of  the  World  War,  of  the  troops  in  Belgium  only 
those  of  the  British  Army  were  adequately  protected.  At  the  beginning 
of  trench  warfare  in  1914  an  epidemic  broke  out,  and  in  January  and 
February  of  1915,  4000  cases  occurred  in  Dunkirk.  Up  to  May  of  1915 
only  827  cases  were  contributed  from  the  British  Army,  the  bulk  of  the 
cases  came  from  among  the  unvaccinated  Belgian  soldiers  and  civilians. 
Vaccination  was  instituted  in  February,  and  the  epidemic  was  at  an 
end  by  the  middle  of  the  summer.  In  the  early  days  of  the  war  vac- 
cination had  not  been  compulsory  in  the  French  Army,  and  as  the 
result  a  large  number  of  troops  were  victims  of  typhoid  fever.  The 
institution  of  vaccination  completely  altered  the  picture.  Courmont 
gives  the  following  statistics  for  the  French  Army  in  1916: 

Deaths 

Non-vaccinated  cases  17.4  per  cent. 

Of  the  vaccinated  cases: 

Those  who  had  one  injection 6.0  per  cent. 

Those  who  had  two  injections 4.0  per  cent 

Those  who  had  three  injections 2.5  per  cent. 

Those  who  had  four  injections 1.9  per  cent. 

Duration  of  Protection. — When  typhoid  vaccination  was  first  in- 
troduced it  was  generally  assumed  that  protection  lasts  for  about  two 
years.  Certain  British  troops  in  Mudros  were  found  to  have  developed 
typhoid  fever  within  six  months  after  inoculation.  Similarly,  certain 
troops  of  the  American  Army  developed  typhoid  fever  a  few  months 
after  they  had  been  vaccinated,  but  it  was  found  upon  investigation 
that  in  this  instance  the  vaccination  had  not  been  completed.  On  the 
basis  of  experience,  yearly  vaccinations  were  practiced  in  the  British 
Army,  although  it  was  not  considered  necessary  to  give  the  three 
doses  at  the  time  of  revaccination ;  a  single  maximum  dose  on  revac- 
cination  apparently  served  to  maintain  immunity.  Yearly  revaccina- 
tion, however,  provides  adequate  protection.  Knowing  that  infection 
has  occurred  within  a  few  months  after  proper  vaccination  it  is  no 
longer  advisable  to  state  that  protection  lasts  for  more  than  a  year. 
The  determination  as  to  when  revaccination  must  be  practiced  depends 
in  certain  measure  upon  the  degree  of  exposure  to  the  disease.  In 
those  districts  where  typhoid  or  paratyphoid  fevers  are  endemic,  we  rec- 
ommend that  vaccination  be  reinforced  by  a  single  yearly  inoculation  of 
the  maximum  dose.  If  a  period  of  two  years  has  elapsed  since  previous 
vaccination,  it  is  advisable  to  revaccinate  with  three  injections. 

Complications. — The  reaction  to  any  dose  of  typhoid  vaccine  is 
extremely  variable.  Usually  the  second  and  third  doses  produce  some- 
what more  severe  reactions  than  the  first  dose.  There  are,  however, 
certain  individuals  who  are  apparently  hypersusceptible  to  typhoid  pro- 
tein, and  these  may  react  with  great  severity.  As  a  rule,  reactions  are 
merely  local  and  are  exhibited  by  swelling,  redness,  tenderness  and  pain 
about  the  site  of  inoculation.  General  reactions  are  much  less  fre- 


PROPHYLACTIC  VACCINATION  291 

quent  and  are  exhibited  by  malaise,  headache,  fever,  constipation  and 
occasionally  chills.  Maurange  investigated  the  general  reaction  fol- 
lowing 39,215  inoculations  and  reports  the  following  results: 

T^mAc  nf  rpaotinn  Typhoid  Para  bacilli 

Types  per  cent.  per  cent. 

None    92.23  98.59 

Feeble    6.18  1.41 

Moderate    1.40  o.oo 

Pronounced    0.19  o.oo 

Rarely  the  inoculations  may  be  followed  by  arthritis,  nephritis  and 
severe  intestinal  disturbances.  Chantemesse  has  called  attention  to  the 
recrudescence  of  tuberculosis  during  immunization,  and  it  has  further 
been  suggested  that  other  chronic  diseases,  including  syphilis,  may  be 
excited  to  renewed  activity.  We  have  observed  cardiac  arrhythmia  in 
individuals  who  have  previously  suffered  from  myocardial  disease. 

Contraindications. — The  contraindications  include  kidney  disease, 
diabetes,  myocardial  and  endocardial  disease,  aortitis,  cachexia,  gastro- 
intestinal disturbances  and  alcoholism.  The  presence  of  acute  infec- 
tions is  also'  regarded  as  contraindicating  vaccination.  According  to 
Maurange,  age  is  no  contraindication,  although  the  relative  unsuscep- 
tibility  of  old  people  to  typhoid  fever  may  make  it  seem  unnecessary  to 
vaccinate.  Menstruation  is  not  a  contraindication. 

Vaccination  Against  Cholera. — This  was  first  employed  by  Ferran 
in  1884.  Haffkine  published  results  in  1888,  and  since  then  numerous 
investigations  have  developed  technical  methods  and  have  emphasized 
the  value  of  protective  vaccination.  Ferran  injected  broth  cultures  of 
living  vibrios  subcutaneously,  employing  0.25  c.c.  as  the  first  dose  and 
0.5  c.c.  as  the  second  and  third  doses.  Haffkine  employed  two  kinds 
of  vaccine,  a  weaker  vaccine  prepared  from  living  organisms  grown 
on  agar  at  39°  C.  and  a  more  virulent  vaccine  prepared  from  vibrios 
which  had  been  passed  through  a  series  of  guinea-pigs.  Subsequently 
Kolle  prepared  a  vaccine  made  from  heat-killed  agar  cultures  of  viru- 
lent organisms.  The  emulsion  is  made  by  suspending  2  mg.  organisms 
in  saline  and  heating  to  60°  C.  for  one  hour.  Cantacuzene  prepared  a 
vaccine  by  heating  emulsions  of  the  vibrios  for  one  and  one-half  hours 
at  55°  to  56°  C.  The  concentration  of  this  vaccine  was  500  to  1000 
million  organisms  per  cubic  centimeter.  Two  inoculations  were  given, 
the  first  of  2.  c.c.  and  the  second  of  4.  c.c.  with  a  six-day  interval. 
Strong  prepared  a  vaccine  by  incubating  the  emulsion  in  sterile  water, 
thereby  breaking  up  the  cells.  The  emulsion  was  then  passed  through 
a  Reichel  filter  and  the  sterile  filtrate  employed.  At  the  present  day 
heat-killed  vaccines  are  most  commonly  employed. 

Results. — The  earlier  work  of  Ferran  and  of  Haffkine  was  ex- 
tremely encouraging,  but  the  subsequent  statistics  lend  even  greater 
support  to  the  value  of  this  procedure.  Arnaud  made  a  study  of 
108,000  men  during  the  second  Balkan  War.  These  men  were  all  in 
infected  areas.  Among  the  unvaccinated  men  the  morbidity  was  5.75 
per  cent.  Among  those  who  had  received  insufficient  vaccination  it  was 
3.12  per  cent.,  and  among  those  who  had  received  the  full  treatment  it 


292  THE  PRINCIPLES  OF  IMMUNOLOGY 

was  0.41  per  cent.  Kobe  made  an  extensive  study  of  the  population 
of  Tokio  and  its  suburbs.  In  the  city  of  Tokio,  10.54  per  cent,  of  the 
entire  population  were  vaccinated.  The  absolute  number  that  were 
vaccinated,  namely,  238,936  in  Tokio  and  61,988  in  the  suburbs,  as  well 
as  the  large  number  of  controls,  provides  a  sufficient  number  from 
which  to  draw  satisfactory  conclusions.  In  Tokio  cholera  occurred  in 
1.85  per  10,000  of  the  unvaccinated  and  0.13  per  10,000  among  the  vac- 
cinated. In  the  suburbs  cholera  occurred  in  3.09  per  10,000  of  the 
unvaccinated,  and  there  were  no  cases  reported  among  the  vaccinated. 
Cantacuzene  has  studied  results  obtained  in  the  campaigns  in  the  Orient 
during  the  Balkan  Wars  and  the  World  War.  These  were  conducted 
particularly  during  the  epidemics,  and  by  a  study  of  the  normal  curve 
of  epidemics  he  finds  that  vaccination  leads  to  a  sharp  drop  in  the 
epidemic  curve  and  incidence  of  the  disease. 

Vaccination  Against  Pneumonia. — Although  vaccination  against 
pneumonia  was  practiced  by  Wright  before  the  various  types  of  pneu- 
mococci  had  been  identified,  it  was  not  until  the  types  were  carefully 
studied  that  exact  results  could  be  obtained.  Lister,  after  he  had  iden- 
tified the  types  of  organisms  present  in  South  Africa,  carried  out 
prophylactic  immunization  in  11,000  workers  in  the  Rand  mines.  He 
employed  a  composite  vaccine  prepared  from  the  pneumococcus  types 
prevalent  in  that  region.  He  found  that  subcutaneous  inoculations  were 
sufficient  to  establish  an  immunity,  and  demonstrated  that  the  pro- 
tection was  effective  against  the  particular  type  of  pneumococcus  used 
in  the  vaccines.  He  emphasized  the  importance  of  using  a  large  bulk 
of  organisms  and  considers  that  the  minimum  effective  dose  is  at  least 
6000  million  pneumococci  of  each  group  against  which  protection  is 
sought.  The  work  of  Cecil  and  Austin  and  o<f  Cecil  and  Vaughan  has 
been  of  the  utmost  importance.  Cecil  and  Austin  employed  a  saline 
suspension  of  killed  pneumococci  of  Types  I,  II  and  III.  Three  or 
four  doses  were  given  at  intervals  of  five  to  seven  days.  The  first  dose 
contained  1000  million  of  each  of  the  three  types ;  the  second  contained 
2000  million  of  each  type,  and  the  third  and  fourth  contained  3000 
million  each  of  Types  I  and  II  and  1500  million  Type  III.  At  Camp 
Upton  12,000  troops  were  vaccinated,  and  among  these  only  seventeen 
cases  of  pneumonia  of  all  types  developed,  including  those  due  to 
Type  IV  as  well  as  to  the  streptococcus.  Among  the  20,000  unvac- 
cinated men,  172  cases  were  reported.  At  Camp  Wheeler  a  lipovaccine 
was  employed  containing  10,000  million  each  of  Types  I,  II  and  III  per 
cubic  centimeter,  given  in  one  dose.  Eighty  per  cent,  of  the  total  com- 
mand, or  13,460  men,  were  vaccinated  and  363  cases  of  pneumonia  of  all 
varieties  developed.  The  study  was  difficult  because  of  the  prevailing 
influenza  epidemic.  An  analysis  of  the  records  shows  that  "  there  were 
thirty-two  cases  of  Types  I,  II  and  III  pneumonia  among  the  vaccinated 
four-fifths  of  camp,  and  forty-two  cases  of  pneumonia  of  these  types 
among  the  unvaccinated  one-fifth  of  camp.  If,  however,  all  cases  of 
pneumonia  that  developed  within  one  week  after  vaccination  are  ex- 
cluded from  the  vaccinated  group,  there  remain  only  eight  cases  of 


PROPHYLACTIC  VACCINATION  293 

pneumonia  produced  by  fixed  types,  and  these  were  all  secondary  to 
severe  attacks  of  influenza.  This  exclusion  is  justified  by  the  fact  that 
protective  bodies  do  not  begin  to  appear  in  the  serum  until  the  eighth 
day  after  injection  of  pneumococcus  lipovaccine."  "  The  pneumonia 
incidence  rate  per  1000  men  during  the  period  of  the  experiment  was 
twice  as  high  for  unvaccinated  recruits  as  for  vaccinated  recruits,  and 
nearly  seven  times  as  high  for  unvaccinated  seasoned  men  as  for 
vaccinated  seasoned  men."  The  death  rate  for  vaccinated  men,  in  whom 
the  pneumonia  developed  more  than  one  week  after  vaccination  was 
12.2  per  cent.,  whereas  among  the  unvaccinated  troops  it  was  22.3  per 
cent.  The  death  rate  for  primary  pneumonias  wasi  only  one-third  as 
great  among  vaccinated  men  as  among  unvaccinated,  but  the  rate  in 
pneumonia  secondary  to  influenza  was  about  the  same  for  both  groups. 
Among  the  20  per  cent,  of  the  command  which  were  unvaccinated  327 
cases  were  reported.  These  statistics  were  sufficiently  encouraging  to 
introduce  vaccination  into  the  army  on  a  fairly  large  scale.  Neverthe- 
less, the  results  are  not  sufficiently  conclusive  to  state  positively  that  a 
high  degree  of  protection  is  obtained.  Recently  Cecil  and  Blake  have 
been  able  to  produce  in  monkeys  a  characteristic  pneumococcus  pneu- 
monia by  intratracheal  inoculation.  These  investigators  have  studied 
the  problem  of  vaccination  with  saline  vaccines  and  lipovaccines  of 
killed  pneumococci  and  in  addition  have  investigated  the  value  of  vac- 
cination with  living  organisms.  They  found  that  vaccination  with  killed 
pneumococci  was  not  effective  in  preventing  the  development  of  the 
disease  under  the  conditions  of  infection  but  that  the  vaccinated  animals 
showed  a  somewhat  less  severe  form  of  the  disease.  The  living  vaccine 
was  considerably  more  satisfactory,  but  they  state  that  "  the  method 
is  too  dangerous  for  any  sort  of  practical  application."  Vaccination 
with  living  virulent  pneumococcus,  Type  I,  produces  a  protective  im- 
munity against  pneumonia  of  homologous  types.  The  immunity  against 
other  types  of  pneumococcus  following  vaccination  with  Type  I  offers  a 
certain  degree  of  protection  against  other  types,  but  this  varies  con- 
siderably with  the  individual  monkey.  Vaccination  with  "  living  aviru- 
lent  pneumococcus  Type  I,  if  administered  in  sufficiently  large  doses, 
renders  the  monkey  immune  to  a  subsequent  pneumonia  of  homologous 
type."  Cecil  and  Blake  point  out  that  "vaccination  with  attenuated 
living  pneumococci  could  probably  be  practiced  with  impunity,  but  the 
problem  of  transporting  and  keeping  alive  large  quantities  of  pneumo- 
cocci in  the  field  would  be  difficult  to  solve." 

Vaccination  Against  Plague. — Prophylactic  vaccination  against 
plague  was  first  reported  by  Haffkine  in  1897.  Subsequently  Pfeiffer 
and  also  Gaffky  reported  experiments  which  support  the  value  of  this 
type  of  vaccination.  Haffkine's  vaccine  was  prepared  from  a  killed  broth 
culture  of  the  bacillus  pestis  five  or  six  weeks  old.  Adult  males  were 
given  3  to  3.5  c.c.  and  adult  females  2  to  2.5  c.c.  Kolle's  vaccine  is 
prepared  from  slant  agar  growths  suspended  in  the  proportion  of  2  mg. 
of  bacilli  to  the  cubic  centimeter  of  salt  solution.  Kolle  and  Strong 
also  employed  living  organisms  whose  virulence  had  been  greatly  re- 


294  THE  PRINCIPLES  OF  IMMUNOLOGY 

duced.  Lustig  and  Galeotti  used  nucleoprotein  extracted  from  the 
organisms  and  Kitano  and  others  have  employed  organisms  grown  in 
Bengal  isinglass  medium.  Kitano  and  Sukegawa  employed  sensitized 
vaccines  and  are  of  the  opinion  that  these  give  better  results  than  the 
usual  heated  vaccine.  They  gave  in  the  first  dose  2  mg.  of  the  sensi- 
tized organism  and  in  the  second  dose  4  mg.  of  the  sensitized  organism. 
If  haste  is  essential,  6  mg.  may  be  given  at  one  dose. 

Experimentally,  it  has  been  established  that  vaccinated  animals  dis- 
play an  increased  resistance  against  the  disease.  The  Indian  Plague 
Commission  reported  that  vaccination  in  man  diminishes  the  incidence 
of  the  disease,  but  that  it  does  not  furnish  absolute  protection.  Appar- 
ently the  duration  of  immunity  lasts  from  a  few  weeks  to  a  few  months, 
but  immune  bodies  are  not  demonstrable  until  ten  days  have  elapsed. 
In  spite  of  the  fact  that  numerous  investigators  have  reported  favorably 
on  vaccination  against  plague,  Flu  has  stated  that  an  analysis  of  the 
statistics  fails  to  furnish  evidence  that  sufficient  attention  has  been 
given  in  the  earlier  studies  to  the  prevalence  of  infected  rats  or  to 
other  hygienic  conditions  which  prevailed. 

Vaccination  Against  Typhus  Fever. — Vaccination  against  this 
disease  has  been  attempted  with  the  serum  of  convalescent  patients,  but 
the  results  have  not  been  highly  satisfactory.  Plotz,  Olitsky  and  Baehr 
employed  a  vaccine  composed  of  fifteen  strains  of  bacillus  typhi  exan- 
thematici.  Of  a  series  of  5251  vaccinated  individuals  where  typhus  was 
epidemic  only  Ihree  contracted  the  disease,  and  in  another  series  of 
8420  cases  only  six  contracted  the  disease.  Although  the  work  of  Plotz 
with  this  organism  has  been  carefully  done  there  is  still  doubt  as  to  its 
exact  etiological  relationship.  In  statistics  concerning  this  disease, 
the  presence  of  infected  lice  should  be  taken  carefully  into  considera- 
tion. It  cannot  be  stated  that  vaccination  in  typhus  has  any  great  value 
until  further  investigations  have  been  conducted. 

Vaccination  Against  Pertussis  (Whooping-Cough). — The  dis- 
covery of  the  bacillus  of  whooping-cough  by  Gengou  almost  immediately 
led  to  investigation  as  to  vaccination.  Luttinger  has  summarized  the 
results  obtained  in  a  large  whooping-cough  clinic  and  by  over  180  private 
physicians  and  health  officers.  The  results  were  sufficiently  encouraging 
to  justify  the  recommendation  of  this  procedure.  Conditions  of  ex- 
posure and  the  nature  of  surroundings,  as  well  as  the  variability  of  the 
disease,  even  in  a  single  epidemic,  makes  the  interpretation  of  statistics 
extremely  difficult. 

Vaccination  Against  Dysentery. — Prophylactic  vaccination  against 
dysentery  has  encountered  great  difficulties  because  of  the  extreme  tox- 
icity  of  the  cultures,  Shiga  attempted  to  overcome  this  by  employing 
mixed  active  and  passive  immunization.  He  used  a  bacterial  vaccine  to 
which  was  added  immune  serum.  Experiments  on  10,000  individuals 
showed  a  definite  decrease  in  the  rate  of  mortality.  Others  have  em- 
ployed toxin-antitoxin  mixtures  with  apparent  success,  but  Hoffmann 
found  that  this  type  of  vaccination  failed  to  have  any  effect  on  the  control 
of  a  dysentery  epidemic  which  he  studied.  Whitmore  and  Fennel  and 


PROPHYLACTIC  VACCINATION  295 

also  Fennel  and  Petersen  have  prepared  lipovaccines.  It  was  found  pos- 
sible to  administer  in  a  single  dose  3000  million  Shiga  organisms,  3200 
million  Y  type  organisms  and  2200  million  Flexner  organisms  without 
marked  local  or  general  reaction.  In  experiments  with  animals  immune 
sera  can  be  prepared  with  much  less  difficulty  when  the  organisms  are 
administered  suspended  in  oil.  The  method  ha&  not  as  yet  been  given 
sufficiently  extensive  trials  in  man  to  justify  definite  'Statements  as  to  its 
efficacy,  but  from  the  experimental  results  obtained  it  appears  to  have 
more  promise  than  any  of  the  other  methods  proposed. 

Vaccination  Against  Influenza. — Vaccination  against  influenza  was 
practiced  very  extensively  in  the  recent  great  epidemic.  The  contro- 
versy over  the  etiological  relationship  of  the  bacillus  of  Pf eiffer  has,  in 
our  opinion,  not  been  settled.  The  results  of  vaccination  with  this 
organism  might  serve  to  settle  in  part  the  question  as  to  the  cause  of 
the  disease,  since  a  high  degree  of  immunity  to  the  disease  following 
vaccination,  if  interpreted  in  the  sense  of  specificity,  would  indicate  that 
the  organism  employed  is  the  exciting  cause.  The  vaccines  which  have 
been  employed  have  been  suspensions  in  salt  solution,  killed  by  heat. 
In  certain  districts  stock  cultures  have  been  employed,  in  others  a  cul- 
ture of  a  strain  or  strains  isolated  during  the  epidemic  has  been  used,  and 
in  still  others  a  mixed  vaccine  has  been  used  composed  of  the  bacillus 
of  Pf  eiffer,  the  streptococcus,  the  pneumococcus,  the  staphylococcus  and 
other  organisms.  Reports  of  striking  success  following  vaccination 
have  been  numerous,  including  in  particular  the  work  of  Duval  and  his 
collaborators.  In  consideration  of  reports  of  this  .sort  the  curve  of  the 
epidemic  has  sometimes  been  overlooked.  Reports  of  certain  other 
investigators  have  not  been  encouraging.  McCoy  states  that  "  the  gen- 
eral impression  gained  from  uncontrolled  use  of  vaccines  is  that  they 
are  of  value  in  the  prevention  of  influenza;  but,  in  every  case  in  which 
vaccines  have  been  tried  under  perfectly-controlled  conditions,  they 
have  failed  to  influence  in  a  definite  manner  either  the  morbidity  or 
the  mortality."  At  best  the  method  must  be  regarded  as  still  in  the 
experimental  stage. 

Vaccination  Against  Other  Diseases. — Vaccines  have  been  pre- 
pared against  scarlatina,  cerebrospinal  meningitis,  tuberculosis  and  con- 
taminated wounds.  Examination  of  the  statistics  presented  fails  to 
produce  convincing  evidence  that  vaccination  against  these  conditions  is 
especially  satisfactory.  As  time  goes  on,  methods  may  be  improved 
and  larger  statistical  evidence  collected. 


APPENDIX  C 
VACCINE  THERAPY 

INTRODUCTION. 

DISEASES  OF  THE  GENITO-URINARY  TRACT. 

GONORRHEA. 

CYSTITIS. 

PYELITIS   AND   SUPPURATIVE   NEPHRITIS. 
DISEASES  OF  THE  SKIN. 

FURUNCULOSIS. 

CARBUNCLES. 

ECZEMA. 

RINGWORM. 

OTHER  SKIN  DISEASES. 
DISEASES  OF  THE  RESPIRATORY  TRACT. 

RHINITIS. 

OZENA. 

ASTHMA. 

PERTUSSIS. 

PNEUMONIA. 

OTHER  DISEASES. 
DISEASES  OF  THE  EYE. 
DISEASES  OF  THE  ALIMENTARY  CANAL. 

TYPHOID  FEVER. 

PARATYPHOID  FEVER. 

DYSENTERY. 
TUBERCULOSIS. 

VACCINE  THERAPY 

Introduction. — A  clear  differentiation  must  be  made  between 
prophylactic  vaccination  and  therapeutic  vaccination.  The  value  of 
various  modes  of  prophylactic  vaccination  has  been  discussed  and  their 
importance  in  protection  against  various  diseases  has  been  outlined. 
For  purposes  of  discussion  of  therapeutic  vaccination  it  is  well  to  con- 
sider the  infectious  diseases  as  either  acute  or  chronic  and  either  local 
or  general.  Acute  infectious  processes  are  for  the  most  part  self- 
limited  and  require  little  in  the  way  of  specific  treatment,  and  spon- 
taneous cure  is  so  regular  as  to  render  difficult  the  interpretation  of 
results  following  therapeutic  vaccination.  Chronic  infectious  diseases 
tend  to  be  progressive  and  finally  result  either  directly  or  indirectly  in 
the  death  of  the  patient.  Statistical  reports  may  show  instances  of 
amelioration  of  the  disease,  but  the  personal  bias  of  the  investigator 
may  sometimes  confuse  the  conclusion.  Generalized  infections  may  be 
treated  by  simple  bacterial  vaccination,  but  the  results  with  sensitized 
vaccines  have  been  better  than  those  with  unsensitized  vaccines.  With 
few  exceptions  the  results  of  therapeutic  vaccination  have  been  best 
in  cases  of  localized  infection.  The  vaccines  employed  may  be  in  the 
form  of  stock  vaccines,  but  the  opinion  is  practically  universal  that 
wherever  possible  the  employment  of  autogenous  vaccines  gives  the 
best  results. 

The  persistence  of  chronic  infections  is,  in  part,  due  to  the  fact  that 
the  chronic  inflammatory  fibrous  tissue  hinders  the  general  absorption 
of  antigenic  materials  produced  by  the  exciting  organism.  Conse- 
296 


VACCINE  THERAPY  297 

quently,  immune  bodies  are  not  produced  in  sufficient  amounts  to 
combat  the  infection.  Vaccination  may  serve  to  stimulate  a  general 
immune  reaction  which  aids  in  the  resistance  to  the  local  lesion.  In 
generalized  infections  the  simple  bacterial  vaccines  may  add  to  the  load 
carried  by  the  body  and  perhaps  reduce  rather  than  enhance  immunity. 
If,  however,  immune  serum  is  added  to  the  vaccine  or  introduced  sep- 
arately, the  serum  may  operate  either  upon  the  body  or  upon  the 
bacteria  so  as  to  favor  resistance. 

DISEASES  OF  THE  GENITO-URINARY  TRACT 

Vaccine  Treatment  of  Gonorrhea. — If  stock  vaccines  are  to  be 
employed,  it  is  desirable  to  use  those  composed  of  a  variety  of  strains 
of  the  organisms.  Many  of  the  vaccines  employed  are  heated  salt  solu- 
tion suspensions  of  the  organisms.  Demonchy  advises  the  use  of  large 
doses  of  unheated  salt  solution  suspensions  of  stock  cultures.  Thomson 
has  prepared  a  so-called  detoxicated  vaccine.  In  the  earlier  method 
Thomson  dissolved  the  organism  in  N/io  NaOH  and  precipitated  with 
N.HC1.  The  toxins  remain  in  the  supernatant  fluid.  Later  he  found  that 
the  toxins  could  be  removed  by  washing  with  0.5  per  cent,  sodium 
acid  phosphate  and  0.5  per  cent,  phenol.  Haworth  employed  sensitized 
vaccine,  and  recently  Sezary  has  recommended  lipovaccine.  Most  in- 
vestigators recommend  the  employment  of  large  doses  of  the  organ- 
isms, ranging  from  a  minimum  of  5000  million  to  a  maximum  of 
25,000  million. 

The  vaccines  have  been  employed  in  acute  gonorrheal  urethritis 
but  with  relatively  little  success.  They  have  also  been  employed  in 
vulvo-vaginitis  in  children,  in  some  instances  with  apparent  success. 
Undoubtedly,  the  field  for  therapeusis  of  this  sort  is  best  realized  in 
gonorrheal  arthritis.  In  this  condition  persistent  vaccination  has  been 
followed  in  many  cases  by  excellent  results.  Somewhat  similar  are  the 
chronic  infections  of  urethral  glands,  prostate,  seminal  vesicles  and  the 
internal  female  genitalia.  Results  from  treatment  of  these  conditions 
warrant  a  trial  of  vaccine  treatment  in  conjunction  with  other  modes 
of  treatment  or  in  those  instances  where  other  forms  of  treatment  have 
failed  or  are  contraindicated. 

Cystitis. — The  organisms  which  may  cause  cystitis  are  variable,  but 
in  those  cases  where  the  disease  is  chronic  and  resistant  to  local  treat- 
ment the  causative  organism  usually  belongs  in  the  colon  typhoid  group, 
the  bacillus  coli  communis  being  the  most  frequent  offender.  In  treat- 
ment of  this  disease  it  is  of  fundamental  importance  to  discover  the 
cause.  In  cases  due  to  the  colon  bacillus  the  vaccine  may  be  given  in 
the  form  of  killed  salt  solution  suspensions  of  organisms  isolated  from 
the  case.  Stock  vaccines  may  be  employed  when  necessary.  The  dose 
is  usually  from  50  million  to  100  million.  Results  have  been  extremely 
variable,  but  the  method  is  sufficiently  well  established  to  justify  trial  in 
resistant  cases.  Of  fundamental  importance  is  the  removal  of  ure- 
thral stricture,  prolapse  of  the  bladder  or  other  local  conditions  which 
retard  cure. 


298  THE  PRINCIPLES  OF  IMMUNOLOGY 

Pyelitis  and  Suppurative  'Nephritis. — In  pyelitis  the  causative  or- 
ganism should  be  discovered  before  vaccine  treatment  is  considered. 
If  due  to  the  bacillus  coli  communis,  autogenous  vaccines  in  doses  of 
from  50  million  to  100  million  organisms  given  at  weekly  intervals 
often  yield  good  results.  Suppurative  nephritis  occasionally  is  improved 
by  vaccination  with  the  causative  organism,  but  the  danger  of  wide- 
spread infection  as  a  result  of  the  disease  is  so  great  that  in  our  opinion 
surgical  measures  are  of  more  immediate  importance  unless  the  general 
condition  of  the  patient  contraindicates  operation. 

DISEASES  OF  THE  SKIN 

Many  of  the  diseases  of  the  skin  and  of  the  subcutaneous  tissues 
depend  upon  the  local  action  of  bacteria ;  a  considerable  number  of  these 
is  susceptible  to  vaccine  treatment.  A  greater  number  of  skin  diseases 
is  the  result  of  more  deep  seated  disorders  and  under  these  circumstances 
it  is  essential  that  the  cause  be  corrected;  in  these  instances  vaccine 
treatment  is  of  little  avail  unless  the  primary  disease  is  one  susceptible  to 
that  mode  of  treatment. 

Furunculosis. — Furuncles  are  usually  caused  by  some  variety  of  the 
staphylococcus,  most  frequently  the  staphylococcus  pyogenes  aureus. 
Occasionally,  furuncles  may  be  the  result  of  streptococcus  infections 
or  of  mixed  infections.  The  single  furuncle  usually  heals  after  the 
pus  is  discharged,  either  naturally  or  surgically,  and  may  clear  up  with- 
out any  interference  whatever.  Patients  are  seen,  however,  in  whom 
furuncles  appear  repeatedly.  In  some  of  these  cases  the  underlying 
cause  is  diabetes  mellitus  and  in  others  it  is  apparently  due  to  a  pro- 
longed decrease  in  the  number  of  circulating  leucocytes.  Vaccination 
in  cases  in  which  the  boils  are  persistent  and  frequent  is  usually  effec- 
tive. Stock  vaccines  are  frequently  employed,  but  in  this  condition,  as 
in  others,  autogenous  vaccines  are  to  be  preferred.  Stock  vaccines  have 
frequently  failed  because  of  failure  to  identify  the  exact  organism 
causing  the  condition.  For  example,  staphylococcus  aureus  stock  vac- 
cines are  employed  on  the  assumption  that  the  boils  are  due  to  this 
organism,  whereas  if  cultures  were  made  from  the  boil  another  organism 
might  be  isolated.  It  is  generally  recommended  that  the  vaccine  be 
composed  of  2000  million  organisms  per  cubic  centimeter.  It  is  im- 
portant that  the  first  dose  be  relatively  small  and  the  increase  in  doses 
gradual.  At  the  first  dose  o.i  c.c.  is  given  and  at  the  second  dose  0.2 
c.c.  is  given  and  the  doses  increase  by  gradations  of  o.i  c.c.  until  the 
maximum  dose  o>f  i.o  c.c.  is  reached,  It  is  often  recommended  that 
the  doses  be  given  eight  days  apart,  but  this  period  may  be  reduced 
with  advantage  to  three  or  four  days.  In  case  of  diabetes  the  vac- 
cination should  proceed  more  slowly  and  with  somewhat  smaller 
doses  than  in  other  cases.  With  the  dosage  recommended,  local 
reactions  are  slight  and  general  reactions  very  rarely  appear.  Vac- 
cination in  furunculosis  usually  gives  excellent  results  and  is  to  be 
highly  recommended. 

Carbuncles. — These  are  also  benefited  in  certain  instances  by  vac- 


VACCINE  THERAPY  299 

cine  treatment,  but  it  must  be  expected  that  many  cases  will  fail  to 
improve.  On  the  whole,  surgical  treatment  is  more  satisfactory. 

Eczema. — The  recent  studies  of  this  disease  have  shown  that  many 
cases  are  the  result  of  hypersusceptibility  to  proteins,  usually  those 
contained  in  food.  Granted  that  such  hypersusceptibility  is  demon- 
strable, treatment  is  in  the  form  of  immunization  to  the  particular 
protein  concerned.  Such  immunization  is  similar  to  that  employed 
in  hay  fever  and  has  been  commented  on  in  the  chapter  on  hyper- 
susceptibility (page  231).  Kolmer  states  that  the  prolonged  admin- 
istration of  an  autogenous  bacterial  vaccine  composed  of  staphylococci 
procured  from  the  scales  or  serous  exudate  has  occasionally  aided  in 
the  treatment  of  obstinate  cases  of  eczema. 

Ringworm. — Strickler  has  recently  employed  a  vaccine  made  of 
several  strains  of  the  fungus  and  is  of  the  opinion  that  the  method  has 
some  value  in  obstinate  cases. 

Other  Skin  Diseases. — Vaccination  has  been  employed  with  a  vari- 
able degree  of  success  in  the  different  f o<rms  of  acne,  sycosis,  scrofulo- 
derma,  impetigo  and  certain  forms  of  erythema, 

DISEASES  OF  THE  RESPIRATORY  TRACT 

Rhinitis. — Vaccination  against  acute  rhinitis  has  been  largely 
prophylactic  in  nature,  and  the  results  of  these  vaccinations  have  been 
in  a  general  way  favorable.  The  exact  cause  of  this  disease  has  not 
been  finally  proven,  but  the  work  of  Foster  indicates  rather  strongly 
that  the  agent  is  a  filterable  virus.  The  prophylactic  vaccines,  however, 
have  been  mixed  stock  vaccines  of  a  variety  of  bacteria,  and  it  seems 
probable  to  us  that  any  success  obtained  upon  this  basis  is  probably 
non-specific.  Coates  is  of  the  opinion  that  if  acute  rhinitis  is  treated 
early  with  vaccines  there  is  likely  to  be  improvement.  The  course  of 
acute  rhinitis  is  so  variable  that  statistical  results  are  open  to  some 
question.  In  chronic  rhinitis  it  is  maintained  that  autogenous  vaccines 
are  of  value.  It  must  be  understood,  however,  that  contributory  causes, 
such  as  adenoids,  enlarged  tonsils,  polyps  and  nasal  deformities  must 
be  removed.  So  much  benefit  accrues  from  the  correcting  of  the 
contributory  causes  that  the  beneficial  effects  of  vaccination  probably 
depend  in  certain  part  upon  the  personal  equation  of  the  observer. 

Ozena. — The  cause  of  this  disease  is  at  present  a  matter  of  con- 
siderable dispute,  and  the  value  of  vaccination  is  undecided.  The  vac- 
cines that  have  been  employed  are  usually  made  from  the  bacillus 
ozense  fetidae  of  Perez.  Horn  claims  that  this  organism  is  similar  to 
the  bacillus  bronchisepticus  and  has  made  polyvalent  stock  vaccines 
which  he  claims  are  highly  successful.  Friel  reports  excellent  results 
from  the  intravenous  administration  of  sensitized  living  vaccine  of 
Friedlander  bacillus.  Ersner  has  had  disappointing  results.  While  im- 
provement may  occur  in  a  certain  percentage  of  cases,  McKenzie  found 
a  marked  tendency  to  relapse  following  the  cessation  of  treatment. 

Asthma. — As  with  eczema,  the  recent  investigations  of  hyper- 
susceptibility have  placed  the  study  of  asthma  upon  an  entirely  new 


300  THE  PRINCIPLES  OF  IMMUNOLOGY 

basis.  According  to  the  work  of  Walker  and  his  collaborators,  certain 
of  the  cases  are  due  to  specific  bacterial  invasion,  and  probably  con- 
tributed to  by  a  certain  degree  of  hypersusceptibility  to  the  organism. 
Bacterial  vaccination  in  these  cases  has  been  accompanied  by  good 
results.  A  complete  investigation  of  the  nature  of  the  case  is  essential 
before  any  form  of  vaccine  treatment  should  be  attempted.  Earlier 
investigators  have  employed  mixed  vaccines  made  of  organisms  obtained 
from  the  sputum. 

Pertussis. — Prophylactic  vaccination  against  this  disease  has  been 
discussed  (page  294).  Therapeutic  vaccination  has  been  employed  by  a 
number  of  workers  with,  in  many  instances,  apparently  favorable  re- 
sults. Luttinger  found  that  in  a  series  of  952  cases  treated  by  vaccina- 
tion the  paroxysmal  stage  averaged  about  thirty-seven  days,  whereas 
149  cases  not  treated  with  vaccine  had  a  duration  of  over  fifty  days. 
Blum  and  Smith  found  that  non-specific  vaccination  was  practically  as 
effective  as  vaccination  with  the  bacillus  of  Gengou.  Barenberg  also 
finds  that  pertussis  vaccine,  even  when  given  in  large  doses,  has  neither 
curative  nor  ameliorating  effect.  Kraus  and  others  have  reported  good 
results  by  the  use  of  a  vaccine  prepared  from  the  sputum.  The  sputum 
is  washed,  mixed  with  ether,  shaken  for  three  or  four  days,  the  ether 
evaporated,  the  mixture  tested  for  sterility  and  given  in  doses  of  i.o  c.c. 
every  three  or  four  days.  If  stock  vaccines  of  the  organisms  are  em- 
ployed, it  is  of  the  utmost  importance  that  they  be  fresh.  Doses  of 
25  million  organisms  may  safely  be  given. 

Pneumonia. — Prophylactic  vaccination  (page  292)  is  distinctly 
more  promising  than  therapeutic  vaccination.  Treatment  with  immune 
serum  (page  256)  is  also  more  promising  than  vaccination.  Coleman 
is  of  the  opinion  that  vaccines  in  pneumonia  are  never  harmful  and 
may  be  beneficial.  Teale  and  Embleton  believe  that  they  have  obtained 
good  results  in  certain  cases.  Shera  expresses  the  belief  that  the  local 
infection  is  too  massive  to  permit  of  vaccine  having  any  appreciable 
effect  in  the  stage  of  consolidation.  The  frequent  occurrence  of  pneu- 
mococcus  septicemia  as  a  part  of  the  disease  makes  it  unlikely  that 
vaccination  will  be  helpful.  In  delayed  resolution  vaccines  are  of  value 
in  some  cases.  Shera  also  states  that  empyemia  when  it  has  reached  the 
chronic  stage  may  be  benefited  by  specific  vaccination. 

Other  Diseases. — Certain  diseases  of  the  accessory  regions  of  the 
respiratory  tract,  including  chronic  median  otitis  and  mastoiditis,  have 
been  treated  by  vaccination  with  the  organism  concerned.  Results 
have  been  variable,  but  inasmuch  as  these  represent  somewhat  isolated 
local  infections  it  is  reasonable  to  attempt  vaccination  in  addition  to 
the  usual  modes  of  treatment. 

DISEASES  OF  THE  EYE 

When  conjunctivitis  becomes  chronic,  specific  vaccination  sometimes 
leads  to  improvement.  The  infecting  agents  include  staphylococcus, 
streptococcus,  bacillus  pyocyaneous,  Friedlander's  bacillus  and  others. 
Autogenous  vaccines  may  be  employed  in  addition  to  other  modes  of 


VACCINE  THERAPY  301 

treatment.  Chronic  conjunctivitis,  due  to  the  Morax-Axenfeld  diplo- 
bacillus,  is  said  to  respond  very  well  to  vaccine  treatment.  In  acute 
pneumococcus  and  gonococcus  conjunctivitis,  especially  with  ulcer, 
Allen  advises  early  and  vigorous  vaccine  therapy  and  reports  good 
results.  It  is  also  stated  that  ophthalmia  neonatorum  sometimes  im- 
proves rapidly  under  vaccine  treatment.  In  none  of  these  conditions, 
however,  is  it  wise  to  neglect  other  forms  of  treatment. 

DISEASES  OF  THE  ALIMENTARY  CANAL 

Typhoid  Fever. — Prophylactic  vaccination  has  unquestioned  value 
in  the  prevention  of  typhoid  fever  (page  285).  Specific  therapeutic 
vaccination  has  been  the  subject  of  experiment  since  the  work  of 
Fraenkel  in  1893.  The  vaccines  employed  have  been  usually  killed 
organisms  either  untreated  or  sensitized,  administered  either  subcu- 
taneously or  intravenously.  Certain  authors  have  also  reported  the  use 
of  living  organisms,  but  this  method  has  not  been  adopted.  Gay,  in  his 
book,  "  Typhoid  Fever,"  reports  the  following  summary  of  results 
obtained  by  various  methods : 

SUMMARY  OF  RESULTS  OBTAINED  BY  RECENT  OBSERVERS  (1913^-1917)  IN  THE  TREAT- 
MENT OF  TYPHOID  FEVER  BY  VACCINES  ADMINISTERED  IN  VARIOUS  WAYS. 


Untreated  vaccine  subcutaneously  .  . 
Sensitized  vaccine  subcutaneously  .  . 
Untreated  vaccine  intravenously 

rv.                  Total    Estimates      Bene-        Mortal- 
Observers     cases       basedon         fited             ity 

.  .     30        iooi        512        46%        14.5% 
..     14          593        239        69%          8.0% 

22              501           233           62%            13  O% 

Sensitized  vaccine  intravenouslv  . 

12             487           31  6           8^%            11.0% 

It  is  usually  stated  that  typhoid  fever  has  a  mortality  of  about  10  per 
cent.,  although  in  the  American  Civil  War  it  exceeded  35  per  cent,  and 
in  the  Franco-Prussian,  Spanish-American  and  Boer  Wars  it  ranged 
between  8  and  14  per  cent.  The  severity  of  epidemics  varies  consid- 
erably, but  at  the  best  there  is  little  in  the  way  of  encouragement  to  be 
found  in  the  table  given  above.  The  basis  upon  which  improvement 
is  estimated  varies  considerably  with  the  different  investigators  and 
the  figures  are  "  distinctly  affected  by  subjective  influences."  Gay  has 
employed  a  sensitized  vaccine  administered  intravenously  and  his  re- 
sults in  ninety-eight  cases  are  summarized  as  follows : 

SUMMARY  OF  RESULTS  IN  NINETY-EIGHT  CASES  OF  TYPHOID  TREATED  BY  INTRA- 
VENOUS INJECTION  OF  SENSITIZED  VACCINE  SEDIMENT. 

No. 

of 

cases 

Aborted  33 

Benefited  32 

Unaffected    ...     33 

The  most  significant  figures  in  this  table  refer  to  those  cases  which 
were  aborted.  Careful  study  of  various  epidemics  fails  to  show  any 
instance  where  such  a  large  percentage  of  the  cases  have  aborted,  and 
it  therefore  seems  probable  that  the  vaccination  had  some  distinct  value. 


Widal 
titer 

Blood 

Treatment 

No.  of 

Per- 

Days 

Age 

on 

culture 

begun 

treat- 

manent 

of 

beginning 

positive 

day 

ments 

normal 

treat- 

treatment 

ment 

26.2 

296.0 

36.6% 

134 

1.88 

20.4 

7.0 

24.2 

156.5 

70-9% 

14.8 

3.20 

30-6 

15-8 

28.8 

II4.8 

84.8% 

13-7 

4-85 

46.8 

33-1 

302  THE  PRINCIPLES  OF  IMMUNOLOGY 

This  rapid  improvement  appeared  to  be  somewhat  more  striking  in  the 
moderate  and  mild  cases  than  in  those  which  were  considered  severe. 

Other  investigators  have  noted  that  non-specific  therapy  has  been 
quite  as  effective  as  the  use  of  specific  typhoid  vaccine.  Kraus  found 
that  colon  bacilli  were  equally  effective  and  others  have  confirmed  this 
observation.  Liidke  has  employed  deutero-albumose,  Weichardt  al- 
bumin solutions,  Nolf  pepton  and  still  others  have  employed  such  sub- 
stances as  dextrose,  colloidal  gold  and  even  normal  salt  solution.  Gay 
admits  that  the  non-specific  form  of  therapy  has  been  as  effective  as 
the  use  of  sensitized  typhoid  vaccines,  but  urges  the  employment  of 
typhoid  vaccine  because  it  may  be  kept  indefinitely  in  dried  form  under 
conditions  of  .strict  asepsis  and  can  readily  be  injected  in  exact  amounts. 
He  further  states  that  "  typhoid  vaccine  has  the  advantage  over  other 
protein  preparations  of  building  up  the  active  immunity  of  the  patient, 
and  a  sensitized  vaccine  will,  in  our  experience,  produce  a  higher  grade 
of  leucocytosis." 

Paratyphoid  Fever. — Rathery  and  others  have  used  therapeutic 
vaccination  in  paratyphoid  B  fever.  It  was  concluded  that  the  treatment 
is  useful,  always  improves  general  condition,  often  shortens  the  fever 
and  has  never  led  to  harmful  results.  Others  have  found  that  typhoid 
vaccine  is  as  effective  in  paratyphoid  as  in  true  typhoid  fever  and  the 
non-specific  therapy  indicated  above  has  also  been  effective. 

Dysentery. — The  vaccine  treatment  of  dysentery  is  confined  to  the 
bacillary  form  and  of  these  varieties  the  cases  due  to  the  Flexner 
bacillus  and  other  related  forms  appear  to  do  much  better  than  those 
caused  by  the  Shiga  bacillus.  Nolf,  from  his  observations  in  the  Belgian 
Army,  concludes  that  vaccine  therapy,  when  administered  by  the  intra- 
venous route  is  the  most  effective  therapeutic  procedure  in  the  more 
chronic  forms  of  bacillary  dysentery.  His  cases  did  not  include  those 
caused  by  the  Shiga  bacillus.  Similar  results  had  been  reported  by 
Baroni  in  the  Roumanian  Army.  He  employed  either  six  injections  of 
killed  organisms  or  four  injections  of  living  vaccine.  Kountze  found 
that  in  typical  cases  of  dysentery,  vaccination  produced  immediate  gen- 
eral improvement  and  reduction  in  the  number  of  stools.  The  study  of 
the  therapy  of  this  disease  has  been  somewhat  hampered  by  the  failure  of 
investigators  to  identify  the  strains  of  organisms  concerned.  Although 
the  results  with  vaccination  have  been  encouraging,  it  is  by  no  means  posi- 
tively proven  that  this  mode  of  treatment  is  superior  to  serum  treatment. 

TUBERCULOSIS 

The  various  forms  of  tuberculin  are  vaccines  and  treatment  by  their 
use  is  an  example  of  vaccine  therapy.  The  methods  of  preparation  of 
the  various  tuberculins  have  been  discussed  (page  238).  Koch's  first 
work  with  tuberculins  was  stimulated  by  the  hope  that  treatment  with 
them  might  be  effective.  The  use  of  the  material  in  larger  amounts 
than  now  seem  necessary  led  to  severe  reactions  on  the  part  of  the 
patients  which  in  some  instances  were  disastrous.  For  many  years 
tuberculin  therapy  was  considered  extremely  dangerous  and  was  prac- 
ticed by  very  few  clinicians.  Recently,  however,  a  more  thorough 


VACCINE  THERAPY  303 

knowledge  of  the  proper  precautions  in  treatment  has  been  built  up  and 
satisfactory  results  are  now  reported.  Applied  to  pulmonary  tubercu- 
losis it  has  been  followed  by  improvement  in  many  cases,  particularly 
in  those  under  sanitarium  treatment.  It  also  is  claimed  to  be  an  im- 
portant aid  in  the  treatment  of  tuberculosis  of  the  bones  and  joints 
and  of  the  eye.  Improvement  has  been  reported  also  in  cases  of  tuber- 
culous enteritis  and  mesenteric  lymphadenitis.  Kleinberg,  however, 
maintains  that  only  a  small  proportion  of  bone  and  joint  cases  improve, 
that  the  majority  show  no  improvement  and  that  in  some  cases  relapses 
occurred  and  new  abscesses  appeared. 

Apparently  the  most  suitable  patients  for  tuberculin  therapy  are 
those  with  incipient  tuberculosis  or  old  cases  of  fibroid  phthisis  with 
fair  or  good  nutrition.  Advanced  or  moderately  advanced  cases  may 
be  so  treated  if  the  general  condition  is  good.  Hamman  and  Wolman 
do  not  consider  marked  general  weakness,  fever,  cardiac  disease, 
nephritis,  epilepsy,  syphilis  of  themselves  contraindications  but  rather 
unfortunate  complications  which  may  prevent  specific  treatment. 

The  injections  are  given  subcutaneously  at  the  lower  angle  of  the 
scapula.  In  order  to  observe  whether  or  not  reaction  occurs  the  in- 
jections are  given  in  the  afternoon  after  the  patient's  temperature  has 
been  taken.  This  avoids  mistaking  an  accidental  afternoon  rise  of  tem- 
perature for  a  rise  due  to  the  tuberculin.  Hamman  and  Wolman  recom- 
mend the  following  range  of  doses: 

Tuberculin  Initial  dose  Maximal  dose 

Old  tuberculin   0.000,000,1  to  0.000,001  c.c.  I.  c.c. 

New  tuberculin  0.000,001      to  0.000,1  c.c.  2.  c.c. 

Bacillus  emulsion    0.000,001      to  0.000,1  c.c.  2.  c.c. 

Three  classes  of  patients  are  recognized:  (i)  children,  (2)  patients 
who  exhibit  a  slight  fever  or  are  not  in  good  condition,  (3)  patients  in 
good  general  condition.  The  smaller  initial  doses  are  for  patients  of 
the  first  two  groups,  the  larger  for  patients  in  the  third  group.  Other 
forms  of  tuberculin  are  employed,  but  the  types  noted  above  have  been 
given  the  most  extensive  trial.  Provided  reactions  are  absent  or  very 
slight,  the  injections  may  be  repeated  every  three  or  four  days.  Tuber- 
culin has  been  given  by  mouth,  but  is  absorbed  irregularly  and  may  pro- 
duce unexpected  reactions.  It  has  also  been  administered  intrafocally 
in  tuberculous  pleurisy,  tuberculous  peritonitis,  lupus  and  tuberculosis 
of  the  joints  and  of  the  tunica  vaginalis.  Results  have  in  some  instances 
been  encouraging.  The  local  reactions  include  pain,  tenderness  and 
swelling.  General  reactions  are  exhibited  by  rise  in  temperature, 
malaise,  headache,  insomnia,  rapid  pulse,  loss  of  weight. 

Shiga  has  recently  reported  upon  the  use  of  a  "  serovaccine."  This 
is  designed  especially  for  prophylactic  injection  in  those  who  by  virtue 
of  family  relations,  constitution  or  other  conditions  are  predisposed  to 
the  disease  and  for  early  incipient  cases.  He  claims  to  have  obtained  ex- 
cellent results  by  weekly  vaccination  with  increasing  amounts  of  the  sero- 
vaccine followed  after  fifteen  injections  by  two  graded  doses  of  living 
avirulent  tubercle  bacilli.  The  method  is  prophylactic  rather  than  curative. 


INDEX 


Abderhalden,    building-stone    theory 

of,  30 
Abrin,  70 

Acquired  immunity,  21 
actively,  22 
artificially,  22 
naturally,  21 
Agglutinability  of  bacteria,  alterations 

of,  92 

Agglutination,  group  reactions,  85 
influence  of  electrolytes  on,  90 
of  heat  on,  89 
of  hydrogen -ion  concentration! 

on,  91 

inhibition  zones  in,  89 
mechanism  of,  91 
physical  basis  of,  93 
Agglutinins,  absorption  of,  86 
bacterial,  79 
immune,  80 

production  of,  80 
macroscopic  titration  of,  83 
main,  80 
major,  80 

microscopic  titration  of,  84 
minor,  80 
nature  of,  92 
normal,  80 
partial,  80 

preliminary  titration  of,  82 
production  of  anti-typhoid,  81 
specificity  of,  85 
Aggressins,  4 

Amboceptor,  activation  of — by  comple- 
ment, 181 
and  complement  in  normal  hemo- 

lysins,  proportions  of,  135 
partial,  123 
Anaphylactic  poisons,  218 

shock,  blood  pressure  in,  214 
.     coagulability  of  blood  in,  215 
distention  of  lungs  in,  213 
ferments  in,  215 
in  guinea-pigs,  212 
in  man,  230 
metabolism  in,  214 
methods  of  preventing,  216 
Anaphylactoid  phenomena,  224 
Anaphylatoxin,  219 
Anaphylaxis,  209 

cellular  theories  of,  220 
cross  reactions  in,  217 
group  reactions  in,  217 
intoxicating  injection  in,  211 
passive,  216 

period  of  incubation  in,  211 
physical  theories  of,  222 
reaction,  212 
sensitization  in,  210 


Anaphylaxis,  specificity  of,  217 

theories  of,  218 

Anthrax,  serum  therapy  of,  259 
Anti-aggressins,  5 
Anti-amboceptors,  136 
Anti-anaphylaxis,  215 
Antibodies,  Bordet  antibody,  179 
leucocyte  antibody,  168 
production  at  site  of  injection,  35 
Antibody,  definition  of,  22 
formation,  site  of,  33 
Anti-complementary     action     of     cells, 

tissue  extracts  and  body-fluids,  183 
Anti-complements,  137 
Anticorps  leucocytaire,  168 
Anti-dysentery    sera,    therapeutic    use 

of,  63 
Antiferment,  248 

determination  in  blood  serum,  249 
Antiferment- ferment  balance,  247 
Antigen,  definition  of,  22 
Anti-rabic  vaccination,  effects  of,  284 
in  man,  284 
results  of,  285 
Antitoxins,  formation  of,  41 

influence  of  temperature  on,  43 
nature  of,  43 
Anti-typhoid  vaccination,  complications 

of,  290 

contraindications  to,  291 
duration  of  protection  in,  290 
method  of,  287 
prophylactic  value  of,  288 
Asthma,  cutaneous  tests  in,  234 

vaccine  treatment  of,  299 
Auto-serum  therapy,  264 
in  syphilis,  265 

Bacteremia,  13 

Bacterial  precipitins,  production  of,  108 

products,  immunization  with,  24 

toxins,  classification  of,  40 

vaccine,  definition  of,  273 
killed,  275 
preparation  of,  275 
Bacteriolysins,  144 
Bacteriolysis,  bioscopic  method  for,  149 

Buxton's  method  for,  148 

in  vitro,  146 

Wright's  method  for,  147 
Bacteriotropins,  162 
Bleeding  a  rabbit,  83 

a  guinea-pig,  127 
Blood  antigen,  117 

groups  classification,  Jansky,  99 
Moss,  100 

serum,  therapeutic  employment  of, 
252 

transfusion,  reactions  to,  105 

305 


306 


INDEX 


Bordet-Gengou  phenomenon,  173 

laboratory    demonstration    of, 

174 
Botulinus  antitoxin,  65 

toxin,  65 
Botulism,  use  of  immune  sera  in,  65 

Canine  distemper,  cutaneous  reaction 

in,  244 

Carbuncles,  vaccine  treatment  in,  298 
Cataphylaxis,  8 
Chemical  agencies,  anti-complementary, 

182 

Chemotaxis,  152 
Cholera,  vaccination  against,  291 

serum  therapy  of,  258 
Cobra  lecithid,  141 
Colloid  shock,  225 
Complement,  alterations  of  amount  of, 

127 

deviation,  148 
distribution  of,  126 
end-piece  of,  133 
fixation,  acid-fast,  205 
delicacy  of,  177 
group  reactions  in,  177 
inhibition  zones  in,  176 
tests,  206 

in  echinococcus  cyst,  207 

in  glanders,  206 

in  gonococcus  infections, 

205  . 

in  malignant  tumors,  207 
in  smallpox,  206 
in  sporotrichosis,  207 
in  syphilis,  186 
in  tuberculosis,  203 
in  typhoid  fever,  206 
in  whooping-cough,  207 
fixing   bodies,    amboceptor,    nature 

of,  180 

relation   of — to   other    im- 
mune bodies,  178 
fractions,  133 
in  hemolysis,  influence  of  amount 

of,  121 

inhibition  of — other  than  by  fixa- 
tion, 182 

method  of  obtaining,  127 
mid-piece  of,  133 
multiplicity  of,  131 
nature  of,  129 
origin  of,  128 
preservation  of,  130 
proportions  of — in  normal  hemoly- 

sins,  135 
titration  of,  119 
variability  of,  131 
Complementary    activity,    influence    of 

lipoids  on,  183 
Complementoids,  132 
Conglutinin,  106,  126 
Crotin,  71 
Curcin,  71 

Cutaneous    reactions,    in    canine    dis- 
temper, 244 


Cutaneous  reactions,  in  glanders,  244 
in  gonococcus  infections,  242 
in  hypersusceptibility,  delicacy 

of,  235 

technic  of,  234 
theories  of,  236 

in  hyphomycetes  infections,  244 
in  leprosy,  244 
in  meningococcus  infections, 

243 

in  pneumococcus  infections,  243 
in  pregnancy,  '244 
in  sporotrichosis,  244 
in  typhoid  fever,  242 
to  vaccine  virus,  243 
Cystitis,  vaccine  treatment  of,  297 
Cytolysins,  115 

organ  specificity  of,  143 
Cytolysis,  summary  of,  149 
Cytotoxins,  autocytotoxins,  144 
heterocytotoxins,  144 
isocytotoxins,  144 
lens,  144 
specificity  of,  142 

Danysz  effect  (or  phenomenon),  50 
Dead  bacteria,  immunization  with,  23 
Defensive  ferments,  245 
Desensitization    in    hypersusceptibility, 

215 
Diphtheria,  active  immunization  against, 

55 

antitoxin,  dosage  of  units  of,  53 
injection    scheme    for    produc- 
tion of,  42 

standardization  of,  44 
technic  of  producing,  42 
therapeutic  use  of,  51 
titration  of,  46 
carriers,    anti-bacterial    serum    in 

treatment  of,  262 
natural  immunity  to,  53 
Schick  test  in,  53 
toxin,  technic  of  producing,  42 
Drug  idiosyncrasies,  237 
Duck-bill  platypus,  76 
Dysentery  antitoxin    (serum  therapy), 

62 

toxin,  62 

vaccination  against,  294 
vaccine  treatment  of,  302 

Eczema,  vaccine  treatment  of,  299 

Eel  serum,  75 

Ehrlich  classification  of  immune  bodies, 

27 

hypothesis,  criticism  of,  28 
side-chain  theory,  26 
objections  to,  49 
Endotoxins,  7 
Erythrocytes,  chemical  agglutination  of, 

105 

fragility  of,  138 
iso-hemagglutinins,  98 
iso-hemolysins,  136 
Esterases,  technic  of  determining,  247 


INDEX 


307 


Exotoxins,  7,  40 

Eye,  diseases  of,  vaccines  in,  300 

Ferment-antiferment  balance,  247 
Ferments,  defensive,  245 

immune,  246 

in  blood,  246 

specificity  of,  245 

Food  adulteration,  detection  of,  113 
Furunculosis,  vaccine  treatment  of,  298 

Gas  gangrene,  prophylactic  use  of  sera 

in,  68 

serum  treatment  of,  67 
use  of  immune  sera  in,  67 
Glanders,  cutaneous  reaction  in,  244 
Gonococcus  infections,  cutaneous  reac- 
tions to,  242 
serum  therapy  of,  263 
vaccine  treatment  of,  297 

Hay  fever,  toxins  in,  23.3 
Hemagglutinins,  98 

auto-hemagglutinins,  98 
hetero-hemagglutinins,  98 
immune   hetero-hemagglutinins,  98 
iso-hemagglutinins,  98 
normal  hetero-hemagglutinins,  98 
Hemolysins,  116 

auto-hemolysins,  116 
bacterial,  139 
collection  of  immune,  118 
immune  hetero-hemolysins,  117 
iso-hemolysins,  136 
preparation  of  immune,  117,  118 
titration  of  immune,  118 
vegetable,  140 
venom,  141 

Hemolysis,  chemical,  139 
group  reactions  in,  123 
physical,  138 
Hemolytic  amboceptor-antigen  union, 

dissociation  of,  122 
and  antigen,  quantitative  rela- 
tions of,  120 
and    complement,    quantitative 

relations  of,  119 
relative  affinities  of,  120 
selective  absorption  of,  12 1 
mechanism  of  operation  of,  125 
nature  of,  124 
rate   of    absorption — by   blood 

cells,  122 
specificity  of,  123 
antigen,  nature  of,  124 

quantitative  relations  of,  120 
complement,   quantitative   relations 

of,  119 

relative  affinities  of,  120 
selective  absorption  of,  121 
Hemophagocytosis,  162 
Hemotoxins,  bacterial,  69 
Hetero-hemolysins,  normal,  134 
Hog-cholera,  serum  therapy  of,  270 
Host  and  parasite,  mutual  relations  of,  I 
factors  favoring,  14 


Hyperleucocytosis,  specific,  170 
Hypersusceptibility,  208 

delicacy  of  cutaneous  tests  in,  235 

natural,  230 

occurrence  of,  208 

technic  of  cutaneous  tests  in,  234 

tests  for,  233 

theories  of  cutaneous  reactions  in, 

236 

Hyphomycetes  infections,  cutaneous  re- 
action in,  244 

Immune  human  serum,  treatment  with, 
266 

reactions,  specificity  of,  29 

sera,  anti-hemolytic  activity  of,  185 
Immunity,  as  result  of  vaccination,  281 

function  of  precipitation  in,  114 

relation  of  anaphylaxis  to,  226 

theories  of  nature  of,  26 

types  of,  16 
Immunization,  active,  15 

passive,  25 
Infections,  primary,  13 

mixed  or  multiple,  13 

secondary,  13 

terminal,  13 

Infection,  production  of,  n 
Infectious  disease,  course  of,  15 

non-specific  therapy  of,  30 
Inflammation,  cellular  participation,  170 

in  resistance,  influence  of,  172 
Influenza,  vaccination  against,  295 
Invader,  entrance  of,  n 

factors  favoring,  13 

inhibiting,  14 
Iso-hemagglutinins,  99 

characters  of,  100 

incidence  of,  100 

in  lower  animals,  101 

mechanism  of,  101 

methods   for  testing  human  blood 
for,  103 

Rous  and  Turner  method,  103 
with  standard  sera,  103 

relation   of — to   blood  transfusion, 

101 
Iso-hemolysins,  normal,  136 

Leprosy,  cutaneous  reaction  in,  244 
Leucocyte  enzymes,  168 

extracts,  for  therapeutic  purposes, 

169 

Leucocytes,  bactericidal  extracts  of,  167 
Leucoprotease,  168 
Leucotoxins,  143 
Lipoyaccines,  277 
Luetin  reaction,  242 
Lymphocyte  ferments,  170 

resistance  to  cancer,  171 

Macrocytase,  173 
Mass  action,  law  of,  50 
Meningococcus  infection,  254 

cutaneous  reaction  to,  243 

serum  therapy  of,  254 
Microcytase,  173 


308 


INDEX 


Natural  hemolysins,  fixation  of  comple- 
ment of,  182 
immunity,  16 

classification  of,  18 

family,  20 

individual,  20 

inherited,  20 

racial,  18 

species,  18 

Neisser-Wechsberg  phenomenon,  147 
Normal  serum,  therapeutic  use  of,  270 

Opsonins,  158 

as  "  facultative  "  amboceptor,  161 
experimental  demonstration  of,  159 
for  cells  other  than  bacteria,  162 
immune,  161 
normal,  159 

specificity  and  other  characters  of, 
163 

Ozena,  vaccine  treatment  of,  299 

Parasite  and  host,  mutual  relations  of,  I 
Parasitic  protozoa,  poisons  of,  75 
Parasitism,  half  parasites,  2 
pure  parasites,  2 

saprophytes,  2 
Paratyphoid  fever,  vaccination  against, 

285 

vaccine  treatment  of,  302 
Pertussis,  vaccination  against,  294 
vaccine  treatment  of,  300 
Pfeiffer  phenomenon,  144 

demonstration  of,  145 
Phagocytic    cells,    influences    operating 

upon,  165 
types  of,  153 
Phagocytosis,  151 

analysis  of  mechanism  of,  165 
digestion  of  bacteria  in,  153 
experimental  demonstration  of,  155 
functions  of,  154 
influence  of  phagocyte  and  ingested 

elements  on,  163 
of  temperature  on,  157 
ingestion  of  foreign  body  in,  153 
mechanism  of,  155 
physical  basis  of,  156 
process  of,  152 

relation  of  bacterial  virulence  to,  164 
surface  tension  in,  156 
Phagolysis,  155 
Phasin,  71 
Phytotoxins,  69 
Plague,  260 

serum  therapy  of,  260 
vaccination  against,  293 
Plant  pollens,  231 
Pneumococcus  infections,  256 

cutaneous  reaction  to,  243 
Pneumonia,  serum  treatment  of,  256 
vaccination  against,  292 
vaccine  treatment  of,  300 
Pneumotoxin,  243 
Poisonous  fish,  75 

substances,  production  of,  6 


Poisons,  bees,  wasps  and  hornets,  74 

centipedes,  74 

duck-bill  platypus,  76 

of  parasitic  protozoa,  75 

scorpion,  74 

spider,  74 

toads,  frogs  and  salamanders,  75 

vegetable,  69 
Poliomyelitis,     acute     anterior,     serum 

therapy  of,  268 
Pollantin,  233 
Precipitation,  106 

biological  relationships  based  on,  in 

delicacy  of,  109 

experimental  demonstration  of,  108 

functions  of — in  immunity,  114 

nature  of  reaction  of,  107 

organ  specificity  of,  112 

physical  basis  of,  109 

practical  application  of,  no 
Precipitin  test  in  game  laws,  1 14 
Pregnancy,  Abderhalden  test,  249 

cutaneous  reaction  in,  244 
Prophylactic  vaccination,  272 
Proteases,  technic  of  determining,  247 
Proteins,  poisonous  bacterial,  7 
Ptomains,  6 

Pyelitis,  vaccine  treatment  of,  298 
Pyemia,  13 

Rabic  vaccine,  preparation  of,  283 
Rabies,  active  immunization  in,  283 

vaccination  against,  282 
Resistance,  factors  operating  against,  14 
Rhinitis,  vaccine  treatment  of,  299 
Ricin,  70 

Rinderpest,  serum  treatment  of,  269 
Ringworm,  vaccine  treatment  of,  299    . 
Robin,  71 

Sapremia,  13 

Septicemia,  13 

Serum,  anti-anthrax,  259 

anti-bacterial,  in  treatment  of  diph- 
theria carriers,  262 

anti-cholera,  258 

anti-gonococcus,  263 

anti-hog-cholera,  270 

anti-meningococcus,  254 

anti-plague,  260 

anti-pneumococcus,  256 

anti-poliomyelitis,  268 

anti-streptococcus,  253 

disease,  accelerated  reaction,  229 

delayed  reaction,  228 
Smallpox  vaccination,  278 

methods  of,  280 

vaccine,  preparation  of,  279 
Sporotrichosis,  cutaneous  reaction  in,  244 
Streptococcus  infections,  serum  therapy 

of,  253 

Suppurative    nephritis,    vaccine    treat- 
ment of,  298 

Syphilis,  auto-serum  therapy  in,  265 
Syphilitic  amboceptor,  190 
nature  of,  191 

antigen,  nature  of,  189 


INDEX 


309 


Tests,  Abderhalden,  249 
Dreyer,  96 
forensic  blood,  no 
tuberculin,  238 
Widal,  85 
Tetanolysin,  57 
Tetanospasmin,  58 
Tetanus,  serum  treatment  of,  60 
antitoxin,  56 

prophylactic  use  of,  59 
therapeutic  use  of,  58 
toxin,  56 

route  of  absorption  of,  58 
Thigmotropism,  171 
Toxin-antitoxin  union,  47 

Ehrlich  theory  of,  47 
Toxins,  bacterial,  38 
gas  bacillus,  67 
general  nature  of,  38 
injury  of,  39 
L+  dose  of,  46 
LQ  dose  of,  46 
minimum  lethal  dose  of,  45 
pathological  effects  of,  41 
pollen,  71 
true,  7 

Tuberculin  reaction,  238 
conjunctival,  239 
cutaneous  reaction  of,  239 
general,  238 
intracutaneous,  239 
percutaneous,  239 
specificity  of,  241 
theories  of,  240 
utility  of,  241 
Tuberculosis,    complement   fixation    in 

203 

serum  treatment  of,  263 
vaccine  treatment  of,  302 
Typhoid   fever,   cutaneous   reaction   in, 

242 

serum  treatment  of,  263 
vaccination  against,  285 
vaccine  treatment  of,  301 
vaccine,  preparation  of,  286 
Typhus  fever,  vaccination  against,  294 

Vaccination,  against  cholera,  291 

dysentery,  294 

influenza,  295 

pertussis,  294 

plague,  293 

pneumonia,  292 

rabies,  282 

typhoid  and  paratyphoid  fevers, 
285 

typhus  fever,  294 
immunity  as  result  of,  281 
unfavorable  results  of,  282 
Vaccine  therapy,  296 

treatment,  in  asthma,  299 

in  carbuncles,  298 

in  cystitis,  297 

in  diseases  of  the  eye,  300 

in  dysentery,  302 


Vaccine  treatment,  in  eczema,  299 
in  furunculosis,  298 
in  gonorrhea,  297 
in  ozena,  299 
in  paratyphoid  fever,  302 
in  pertussis,  300 
in  pneumonia,  300 
in  pyelitis,  298 
in  rhinitis,  299 
in  ringworm,  299 
in  suppurative  nephritis,  298 
in  tuberculosis,  302 
in  typhoid  fever,  301 
virus,  cutaneous  reaction  to,  243 
Vaccines,  definition  of,  273 
dosage  of,  277 
method  of  counting,  276 
types  of,  274 

autogenous,  275 
killed  bacterial,  275 
living,  274 
mixed,  275 
sensitized,  274 
stock,  275 
tetra,  287 
triple,  287 
Vaccinia,  281 
Vaccinoid,  243 

Venom  cobra,  clinical  tests,  142 
Venoms,  pathological  effects  of,  73 
production  of  antisera  for,  73 
snake,  71 
Virulence,  2 

alteration  of,  8 
basis  of,  3 
decrease  of,  9 
demonstration  of,  3 
increase  of,  8 
Virulins,  5 

Wassermann  reaction,  186 

antigen  for,  187 

complement  for,  191 

dependability  of,  202 

diagnostic  value  of,  201 

end-piece  in,  134 

hemolytic  system  for,  194 

influence  of  temperature  upon, 
196 

interpretation  of  results  of,  202 

modifications  of,  200 

post-mortem,  203 

preservation     of     erythrocytes 
for,  195 

quantitative  results  with,  203 

reading,  199 

specificity  of,  200 

on  spinal  fluid,  203 

technic  of,  196 
test,  protocol  of,  199 

table  of  methods  of  perform- 
ing, 192 
Weber-Fechner  law,  152 

Zootoxins,  71 


14  DAY  USE 

RETURN  TO  DESK  FROM  WHICH  BORROWED 

Ea 

This  book  is  due  on  the  last  date  stamped  below,  or 

on  the  date  to  which  renewed. 
Renewed  books  are  subject  to  immediate  recall. 


I 

1AM     5  1964 

u           ;W 

i       JUN  9     1966 

:V,U 

JUN  10  1969 

JUN    719691 

1 

LD  21-50m-4,'63 
(D6471slO)476 


General  Library 

University  of  California 

Berkeley 


LD  21-100ra-7 


